Vitronectin receptor antagonist pharmaceuticals

ABSTRACT

The present invention describes novel compounds of the formula:
 
(Q) d -L n -C h ,
 
useful for the diagnosis and treatment of cancer, methods of imaging tumors in a patient, and methods of treating cancer in a patient. The present invention also provides novel compounds useful for monitoring therapeutic angiogenesis treatment and destruction of new angiogenic vasculature. The present invention further provides novel compounds useful for imaging atherosclerosis, restenosis, cardiac ischemia and myocardial reperfusion injury. The present invention still further provides novel compounds useful for the treatment of rheumatoid arthritis. The pharmaceuticals are comprised of a targeting moiety that binds to a receptor that is upregulated during angiogenesis, an optional linking group, and a therapeutically effective radioisotope or diagnostically effective imageable moiety. The imageable moiety is a gamma ray or positron emitting radioisotope, a magnetic resonance imaging contrast agent, an X-ray contrast agent, or an ultrasound contrast agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/599,365, filed Jun.21, 2000, now U.S. Pat. No. 6,569,402, which is a continuation-in-partof U.S. Ser. No. 09/466,582, filed Dec. 17, 1999, now U.S. Pat. No.6,558,649, which claims the benefit of U.S. Ser. No. 60/112,831, filedDec. 18, 1998, the entire disclosures of which are incorporated hereinin their entireties.

FIELD OF THE INVENTION

The present invention provides novel pharmaceuticals useful for thediagnosis and treatment of cancer, methods of imaging tumors in apatient, and methods of treating cancer in a patient. The invention isalso directed to novel pharmaceutical compositions and combinationtherapy comprising a compound of the invention or a pharmaceuticallyacceptable salt thereof, and at least one agent selected from the groupconsisting of a chemotherapeutic agent and a radiosensitizer agent. Thepharmaceuticals are comprised of a targeting moiety that binds to thevitronectin receptor that is expressed in tumor vasculature, an optionallinking group, and a therapeutically effective radioisotope ordiagnostically effective imageable moiety. The therapeutically effectiveradioisotope emits a gamma ray or alpha particle sufficient to becytotoxic. The imageable moiety is a gamma ray or positron emittingradioisotope, a magnetic resonance imaging contrast agent, an X-raycontrast agent, or an ultrasound contrast agent.

BACKGROUND OF THE INVENTION

Cancer is a major public health concern in the United States and aroundthe world. It is estimated that over 1 million new cases of invasivecancer will be diagnosed in the United States in 1998. The mostprevalent forms of the disease are solid tumors of the lung, breast,prostate, colon and rectum. Cancer is typically diagnosed by acombination of in vitro tests and imaging procedures. The imagingprocedures include X-ray computed tomography, magnetic resonanceimaging, ultrasound imaging and radionuclide scintigraphy. Frequently, acontrast agent is administered to the patient to enhance the imageobtained by X-ray CT, MRI and ultrasound, and the administration of aradiopharmaceutical that localizes in tumors is required forradionuclide scintigraphy.

Treatment of cancer typically involves the use of external beamradiation therapy and chemotherapy, either alone or in combination,depending on the type and extent of the disease. A number ofchemotherapeutic agents are available, but generally they all sufferfrom a lack of specificity for tumors versus normal tissues, resultingin considerable side-effects. The effectiveness of these treatmentmodalities is also limited, as evidenced by the high mortality rates fora number of cancer types, especially the more prevalent solid tumordiseases. More effective and specific treatment means continue to beneeded.

Despite the variety of imaging procedures available for the diagnosis ofcancer, there remains a need for improved methods. In particular,methods that can better differentiate between cancer and otherpathologic conditions or benign physiologic abnormalities are needed.One means of achieving this desired improvement would be to administerto the patient a metallopharmaceutical that localizes specifically inthe tumor by binding to a receptor expressed only in tumors or expressedto a significantly greater extent in tumors than in other tissue. Thelocation of the metallopharmaceutical could then be detected externallyeither by its imageable emission in the case of certainradiopharmaceuticals or by its effect on the relaxation rate of water inthe immediate vicinity in the case of magnetic resonance imagingcontrast agents.

This tumor specific metallopharmaceutical approach can also be used forthe treatment of cancer when the metallopharmaceutical is comprised of aparticle emitting radioisotope. The radioactive decay of the isotope atthe site of the tumor results in sufficient ionizing radiation to betoxic to the tumor cells. The specificity of this approach for tumorsminimizes the amount of normal tissue that is exposed to the cytotoxicagent and thus may provide more effective treatment with fewerside-effects.

Previous efforts to achieve these desired improvements in cancer imagingand treatment have centered on the use of radionuclide labeledmonoclonal antibodies, antibody fragments and other proteins orpolypeptides that bind to tumor cell surface receptors. The specificityof these radiopharmaceuticals is frequently very high, but they sufferfrom several disadvantages. First, because of their high molecularweight, they are generally cleared from the blood stream very slowly,resulting in a prolonged blood background in the images. Also, due totheir molecular weight they do not extravasate readily at the site ofthe tumor and then only slowly diffuse through the extravascular spaceto the tumor cell surface. This results in a very limited amount of theradiopharmaceutical reaching the receptors and thus very low signalintensity in imaging and insufficient cytotoxic effect for treatment.

Alternative approaches to cancer imaging and therapy have involved theuse of small molecules, such as peptides, that bind to tumor cellsurface receptors. An In-111 labeled somatostatin receptor bindingpeptide, In-111-DTPA-D-Phe¹-octeotide, is in clinical use in manycountries for imaging tumors that express the somatostatin receptor(Baker, et al. Life Sci., 1991, 49, 1583–91 and Krenning, et al., Eur.J. Nucl. Med., 1993, 20, 716–31). Higher doses of thisradiopharmaceutical have been investigated for potential treatment ofthese types of cancer (Krenning, et al., Digestion, 1996, 57, 57–61).Several groups are investigating the use of Tc-99m labeled analogs ofIn-111-DTPA-D-Phe¹-octeotide for imaging and Re-186 labeled analogs fortherapy (Flanagan, et al., U.S. Pat. No. 5,556,939, Lyle, et al., U.S.Pat. No. 5,382,654, and Albert et al., U.S. Pat. No. 5,650,134).

Angiogenesis is the process by which new blood vessels are formed frompre-existing capillaries or post capillary venules; it is an importantcomponent of a variety of physiological processes including ovulation,embryonic development, wound repair, and collateral vascular generationin the myocardium. It is also central to a number of pathologicalconditions such as tumor growth and metastasis, diabetic retinopathy,and macular degeneration. The process begins with the activation ofexisting vascular endothelial cells in response to a variety ofcytokines and growth factors. Tumor released cytokines or angiogenicfactors stimulate vascular endothelial cells by interacting withspecific cell surface receptors for the factors. The activatedendothelial cells secrete enzymes that degrade the basement membrane ofthe vessels. The endothelial cells then proliferate and invade into thetumor tissue. The endothelial cells differentiate to form lumens, makingnew vessel offshoots of pre-existing vessels. The new blood vessels thenprovide nutrients to the tumor permitting further growth and a route formetastasis.

Under normal conditions, endothelial cell proliferation is a very slowprocess, but it increases for a short period of time duringembryogenesis, ovulation and wound healing. This temporary increase incell turnover is governed by a combination of a number of growthstimulatory factors and growth suppressing factors. In pathologicalangiogenesis, this normal balance is disrupted resulting in continuedincreased endothelial cell proliferation. Some of the proangiogenicfactors that have been identified include basic fibroblast growth factor(bFGF), angiogenin, TGF-alpha, TGF-beta, and vascular endothelium growthfactor (VEGF). While interferon-alpha, interferon-beta andthrombospondin are examples of angiogenesis suppressors.

The proliferation and migration of endothelial cells in theextracellular matrix is mediated by interaction with a variety of celladhesion molecules (Folkman, J., Nature Medicine, 1995, 1, 27–31).Integrins are a diverse family of heterodimeric cell surface receptorsby which endothelial cells attach to the extracellular matrix, eachother and other cells. The integrin α_(v)β₃ is a receptor for a widevariety for a wide variety of extracellular matrix proteins with anexposed tripeptide Arg-Gly-Asp moiety and mediates cellular adhesion toits ligand: vitronectin, fibronectin, and fibrinogen, among others. Theintegrin α_(v)β₃ is minimally expressed on normal blood vessels, but issignificantly upregulated on vascular cells within a variety of humantumors. The role of the α_(v)β₃ receptors is to mediate the interactionof the endothelial cells and the extracellular matrix and facilitate themigration of the cells in the direction of the angiogenic signal, thetumor cell population. Angiogenesis induced by bFGF or TNF-alpha dependon the agency of the integrin α_(v)β₃, while angiogenesis induced byVEGF depends on the integrin α_(v)β₃ (Cheresh et. al., Science, 1955,270, 1500–2). Induction of expression of the integrins α₁β₂ and α₂β₁ onthe endothelial cell surface is another important mechanism by whichVEGF promotes angiogenesis (Senger, et. al., Proc. Natl. Acad, Sci USA,1997, 84, 13612–7).

Angiogenic factors interact with endothelial cell surface receptors suchas the receptor tyrosine kinases EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1,Tek, tie, neuropilin-1, endoglin, endosialin, and Axl. The receptorsFlk-1/KDR, neuropilin-1, and Flt-1 recognize VEGF and these interactionsplay key roles in VEGF-induced angiogenesis. The Tie subfamily ofreceptor tyrosine kinases are also expressed prominently during bloodvessel formation.

Because of the importance of angiogenesis to tumor growth andmetastasis, a number of chemotherapeutic approaches are being developedto interfere with or prevent this process. One of these approaches,involves the use of anti-angiogenic proteins such as angiostatin andendostatin. Angiostatin is a 38 kDa fragment of plasminogen that hasbeen shown in animal models to be a potent inhibitor of endothelial cellproliferation. (O'Reilly et. al., Cell, 1994, 79, 315–328) Endostatin isa 20 kDa C-terminal fragment of collagen XVIII that has also been shownto be a potent inhibitor. (O'Reilly et. al., Cell, 1997, 88, 277–285)Systemic therapy with endostatin has been shown to result in stronganti-tumor activity in animal models. However, human clinical trials ofthese two chemotherapeutic agents of biological origin have beenhampered by lack of availability.

Another approach to anti-angiogenic therapy is to use targeting moietiesthat interact with endothelial cell surface receptors expressed in theangiogenic vasculature to which are attached chemotherapeutic agents.Burrows and Thorpe (Proc. Nat. Acad. Sci, USA, 1993, 90, 8996–9000)described the use of an antibody-immunotoxin conjugate to eradicatetumors in a mouse model by destroying the tumor vasculature. Theantibody was raised against an endothelial cell class II antigen of themajor histocompatibility complex and was then conjugated with thecytotoxic agent, deglycosylated ricin A chain. The same group (Clin.Can. Res., 1995, 1, 1623–1634) investigated the use of antibodies raisedagainst the endothelial cell surface receptor, endoglin, conjugated todeglycosylated ricin A chain. Both of these conjugates exhibited potentanti-tumor activity in mouse models. However, both still sufferdrawbacks to routine human use. As with most antibodies or other large,foreign proteins, there is considerable risk of immunologic toxicitywhich could limit or preclude administration to humans. Also, while thevasculature targeting may improve the local concentration of theattached chemotherapeutic agents, the agents still must be cleaved fromthe antibody carrier and be transported or diffuse into the cells to becytotoxic.

Thus, it is desirable to provide anti-angiogenic pharmaceuticals andtumor or new vasculature imaging agents which do not suffer from poordiffusion or transportation, possible immunologic toxicity, limitedavailability, and/or a lack of specificity.

There continues to be a need for more effective treatment options forpatients with solid tumors. This is especially true in cases ofmetastatic cancer in which current standard chemotherapy and externalbeam radiation regimens only result in marginal survival improvements.

Although improvements in cytotoxic chemotherapeutics have been made inrecent years, the toxicity of these compounds to normal tissues hascontinued to severely limit their utility in extending survival inpatients with solid tumors. Recently developed combinations of differenttherapeutic modalities, such as external beam irradiation andchemotherapy (i.e. chemoradiation), has provided some incrementalbenefit to the control of tumor progression and quality of life.However, neither systemic chemotherapeutics nor external beamirradiation have acceptable therapeutic indices, and are often limiteddue to unacceptable toxicity to normal tissues. The concept of combinedtherapy of cancer using anti-angiogenesis drugs in combination withchemotherapeutics is not new. Further, the concept of combining targetedin-vivo radiotherapy using radiolabeled antibodies and antibodyfragments with chemotherapy has been reported (Stein R, Juweid M, ZhangC, et al., Clin. Cancer Res., 5: 3199s–3206s, 1999. However, thecombination of a angiogenesis-targeted therapeutic radiopharmaceuticalwhich is targeted to receptors, which are then upregulated in theneovasculature of tumors, together with chemotherapy has not beendescribed before. Therefore, there is a need for a combination of atherapeutic radiopharmaceutical, which is targeted to localize in theneovasculature of tumors, with chemotherapeutics or a radiosensitizeragent, or a pharmaceutically acceptable salt thereof, to provideadditive or synergistic therapeutic response without unacceptableadditive toxicity in the treatment of solid tumors.

The major advantage of combined chemotherapy and angiogenesis-targetedtherapeutic radiopharmaceuticals, over each therapeutic modality alone,is improved tumor response without substantial increases in toxicityover either treatment alone. The advantage of using neovascular-specificradiopharmaceuticals, versus a tumor-cell targeted antibody, is thatthere is much lower systemic radiation exposure to the subject beingtreated.

Further, if the receptor targets for the radiopharmaceutical compounds,used in this method of treatment, are expressed on the luminal side oftumor vessels, there is no requirement that these compounds traverse thecapillary bed and bind to the tumor itself.

Thus, it is desirable to provide a combination of angiogenesis-targetedtherapeutic radiopharmaceuticals and a chemotherapeutics or aradiosensitizer agent, or a pharmaceutically acceptable salt thereof,which target the luminal side of the neovasculature of tumors, toprovide a surprising, and enhanced degree of tumor suppression relativeto each treatment modality alone without significant additive toxicity.

Another application of anti-angiogenic therapy is in treating rheumatoidarthritis (RA). In RA, the ingrowth of a highly vascularized pannus iscaused by the excessive production of angiogenic factors by theinfiltrating macrophages, immune cells, or inflammatory cells.Therefore, it is desirable to have new pharmaceuticals to destroy thehighly vascularized pannus that results and thus treat the disease.

There is also a growing interest in therapeutic angiogenesis to improveblood flow in regions of the body that have become ischemic or poorlyperfused. Several investigators are using growth factors administeredlocally to cause new vasculature to form either in the limbs or theheart. The growth factors VEGF and bFGF are the most common for thisapplication. Recent publications include: Takeshita, S., et. al., J.Clin. Invest., 1994, 93, 662–670; and Schaper, W. and Schaper, J.,Collateral Circulation: Heart, Brain, Kidney, Limbs, Kluwer AcademicPublishers, Boston, 1993. The main applications that are underinvestigation in a number of laboratories are for improving cardiacblood flow and in improving peripheral vessal blood flow in the limbs.For example, Henry, T. et. al. (J. Amer. College Cardiology, 1998, 31,65A) describe the use of recombinant human VEGF in patients forimproving myocardial perfusion by therapeutic angiogenesis. Patientsreceived infusions of rhVEGF and were monitored by nuclear perfusionimaging 30 and 60 days post treatment to determine improvement inmyocardial perfusion. About 50% of patients showed improvement bynuclear perfusion imaging whereas 5/7 showed new collatoralization byangiography. Thus, it is desirable to discover a method of monitoringimproved cardiac blood flow which is targeted to new collatoral vesselsthemselves and not, as in nuclear perfusion imaging, a regionalconsequence of new collatoral vessels.

The detection, imaging and diagnosis of a number of cardiovasculardiseases need to be improved, including restenosis, atherosclerosis,myocardial reperfusion injury, and myocardial ischemia, stunning orinfarction. It has recently been determined that in all of these diseaseconditions, the integrin receptor α_(v)β₃ plays an important role.

For example, in the restenosis complication that occurs in ˜30–50% ofpatients having undergone angioplasty or stent placement, neointimalhyperplasia and ultimate reocclusion is caused by aggressivelyproliferating vascular smooth muscle cells that express α_(v)β₃.(Cardiovascular Res., 1997, 36, 408–428; DDT, 1997, 2, 187–199; CurrentPharm. Design, 1997, 3, 545–584)

Atherosclerosis proceeds from an intial endothelial damage that resultsin the recruitment and subintimal migration of monocytes at the site ofthe injury. Growth factors are released which induce medial smoothmuscle cells to proliferate and migrate to the intimal layer. Themigrating smooth muscle cells express α_(v)β₃.

In reperfusion injury, neutrophil transmigration is integrin dependentand the integrins moderate initial infiltration into the viable borderzone. The induction of α₅β₁, α₄β₁ and α_(v)β₅ in infiltratingneutrophils occurs within 3 to 5 hours after reperfusion as neutrophilsmove from the border zone to the area of necrosis. (Circulation, 1999,100, I-275)

Acute or chronic occlusion of a coronary artery is known to result inangiogenesis in the heart as native collateral vessels are recruited toattempt to relieve the ischemia. However, even a gradual occlusionusually results in areas of infarction as the resulting angiogenesis isnot sufficient to prevent damage. Cardiac angiogenesis has beenassociated with increased expression of the growth factors VEGF and FGFand the upregulation of the growth factor receptors flt-1 and flk-1/KDR.(Drugs, 1999, 58, 391–396)

SUMMARY OF THE INVENTION

It is one object of the present invention to provide improvedanti-angiogenic pharmaceuticals, comprised of a targeting moiety thatbinds to the vitronectin receptor that is expressed in tumorneovasculature, an optional linking group, and a radioisotope. Thevitronectin receptor binding compounds target the radioisotope to thetumor neovasculature. The beta or alpha-particle emitting radioisotopeemits a cytotoxic amount of ionizing radiation which results in celldeath. The penetrating ability of radiation obviates the requirementthat the cytotoxic agent diffuse or be transported into the cell to becytotoxic.

It is another object of the present invention to provide pharmaceuticalsto treat rheumatoid arthritis. These pharmaceuticals comprise atargeting moiety that binds to a receptor that is upregulated duringangiogenesis, an optional linking group, and a radioisotope that emitscytotoxic radiation (i.e., beta particles, alpha particles and Auger orCoster-Kronig electrons). In rheumatoid arthritis, the ingrowth of ahighly vascularized pannus is caused by the excessive production ofangiogenic factors by the infiltrating macrophages, immune cells, orinflammatory cells. Therefore, the radiopharmaceuticals of the presentinvention that emit cytotoxic radiation could be used to destroy the newangiogenic vasculature that results and thus treat the disease.

It is another object of the present invention to provide imaging agents,comprised of vitronectin receptor binding compounds conjugated to animageable moiety, such as a gamma ray or positron emitting radioisotope,a magnetic resonance imaging contrast agent, an X-ray contrast agent, oran ultrasound contrast agent. These imaging agents are useful forimaging tumor neovasculature, therapeutic angiogenesis interventions inthe heart, natural angiogenic processes in response to acute or chroniccoronary vessel occlusion, restenosis post-angioplasty, atherosclerosisand plaque formation, and reperfusion injury.

It is another object of the present invention to provide compoundsuseful for preparing the pharmaceuticals of the present invention. Thesecompounds are comprised of a non-peptide benzodiazepine,benzodiazepinedione, or dibenzotrihydroannulene containing targetingmoiety that binds to a receptor that is upregulated during angiogenesisor during cardiovascular diseases, Q, an optional linking group, L_(n),and a metal chelator or bonding moiety, C_(h). The compounds may haveone or more protecting groups attached to the metal chelator or bondingmoiety. The protecting groups provide improved stability to the reagentsfor long-term storage and are removed either immediately prior to orconcurrent with the synthesis of the radiopharmaceuticals.Alternatively, the compounds of the present invention are comprised of apeptide or peptidomimetic targeting moiety that binds to a receptor thatis upregulated during angiogenesis or during cardiovascular diseases, Q,an optional linking group, L_(n), and a surfactant, S_(f).

The pharmaceuticals of the present invention may be used for diagnosticand/or therapeutic purposes. Diagnostic radiopharmaceuticals of thepresent invention are pharmaceuticals comprised of a diagnosticallyuseful radionuclide (i.e., a radioactive metal ion that has imageablegamma ray or positron emissions). Therapeutic radiopharmaceuticals ofthe present invention are pharmaceuticals comprised of a therapeuticallyuseful radionuclide, a radioactive metal ion that emits ionizingradiation such as beta particles, alpha particles and Auger orCoster-Kronig electrons.

The pharmaceuticals comprising a gamma ray or positron emittingradioactive metal ion are useful for imaging tumors and by gammascintigraphy or positron emission tomography. The pharmaceuticalscomprising a gamma ray or positron emitting radioactive metal ion arealso useful for imaging therapeutic angiogenesis, natural angiogenicprocesses in response to acute or chronic coronary vessel occlusion,restenosis post-angioplasty, atherosclerosis and plaque formation, andreperfusion injury by gamma scintigraphy or positron emissiontomography. The pharmaceuticals comprising a particle emittingradioactive metal ion are useful for treating cancer by delivering acytotoxic dose of radiation to the tumors. The pharmaceuticalscomprising a particle emitting radioactive metal ion are also useful fortreating rheumatoid arthritis by destroying the formation of angiogenicvasculature. The pharmaceuticals comprising a paramagnetic metal ion areuseful as magnetic resonance imaging contrast agents. Thepharmaceuticals comprising one or more X-ray absorbing or “heavy” atomsof atomic number 20 or greater are useful as X-ray contrast agents. Thepharmaceuticals comprising a microbubble of a biocompatible gas, aliquid carrier, and a surfactant microsphere, are useful as ultrasoundcontrast agents.

DETAILED DESCRIPTION OF THE INVENTION

-   [1] Thus, in a first embodiment, the present invention provides a    novel compound, comprising: a targeting moiety and a chelator,    wherein the targeting moiety is bound to the chelator, is a    benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene    nonpeptide, and binds to a receptor that is upregulated during    angiogenesis and the compound has 0–1 linking groups between the    targeting moiety and chelator.-   [2] In a preferred embodiment, the receptor is the integrin α_(v)β₃    or α_(v)β₅ and the compound is of the formula:    (Q)_(d)-L_(n)-C_(h) or (Q)_(d)-L_(n)-(C_(h))_(d′)    wherein, Q is a compound of Formulae (Ia), (Ib) or (Ic):

wherein:

-   R¹ and R³ are independently selected from the group: C₁–C₆ alkyl,    benzyl, phenethyl, and a bond to L_(n); provided that one of R¹ and    R³ is a bond to L_(n);-   R² is independently selected from the group: 2-benzimidazolylmethyl,    2-guanidinoethyl, 2-amino-2-pyridyl, 2-amino-2-pyridylmethyl,    5-amino-2-imidazolylmethyl, and 2-imidazolylmethyl;-   R⁴ is independently selected from H, C₁₋₆ alkyl or benzyl;-   R^(2a) is (CH₂)₃R^(3a);-   R^(3a) is selected from the group:

-   R^(4a) is independently selected from C₁₋₆ alkyl substituted with a    bond to L_(n) or benzyl substituted with a bond to L_(n);-   R^(2b) is independently selected from the group:

the asterisks * denote optional positions for attaching L_(n);

-   or Q is a peptide selected from the group:

-   R^(1p) is L-valine, D-valine or L-lysine optionally substituted on    the • amino group with a bond to L_(n);-   R^(2p) is L-phenylalanine, D-phenylalanine, D-1-naphthylalanine,    2-aminothiazole-4-acetic acid or tyrosine, the tyrosine optionally    substituted on the hydroxy group with a bond to L_(n);-   R^(3p) is D-valine;-   R^(4p) is D-tyrosine substituted on the hydroxy group with a bond to    L_(n);-   provided that one of R^(1p) and R^(2p) in each Q is substituted with    a bond to L_(n), and further provided that when R^(2p) is    2-aminothiazole-4-acetic acid, K is N-methylarginine;-   provided that at least one Q is a compound of Formula Ia Ib, or Ic;-   d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   d′ is 1–100;-   L_(n) is a linking group having the formula:    ((W)_(h)—(CR⁶R⁷)_(g))_(x)-(Z)_(k)-((CR^(6a)R^(7a))_(g′)—(W)_(h′))_(x′);-   W is independently selected at each occurrence from the group: O, S,    NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)NR⁸, C(═O), C(═O)O, OC(═O),    NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′),    (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0–3 R¹⁰, C₃₋₁₀    cycloalkyl substituted with 0–3 R¹⁰, and a 5–10 membered    heterocyclic ring system containing 1–4 heteroatoms independently    selected from N, S, and O and substituted with 0–3 R¹⁰;-   R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at each    occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁–C₅ alkyl    substituted with 0–3 R¹⁰, aryl substituted with 0–3 R¹⁰, benzyl    substituted with 0–3 R¹⁰, and C₁–C₅ alkoxy substituted with 0–3 R¹⁰,    NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to    C_(h);-   R¹⁰ is independently selected at each occurrence from the group: a    bond to C_(h), COOR¹¹, C(═O)NHR¹¹, NHC(═O)R¹¹, OH, NHR¹¹, SO₃H,    PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0–3 R¹¹, C₁₋₅ alkyl    substituted with 0–1 R¹², C₁₋₅ alkoxy substituted with 0–1 R¹², and    a 5–10 membered heterocyclic ring system containing 1–4 heteroatoms    independently selected from N, S, and O and substituted with 0–3    R¹¹;-   R¹¹ is independently selected at each occurrence from the group: H,    alkyl substituted with 0–1 R¹², aryl substituted with 0–1 R¹², a    5–10 membered heterocyclic ring system containing 1–4 heteroatoms    independently selected from N, S, and O and substituted with 0–1    R¹², C₃₋₁₀ cycloalkyl substituted with 0–1 R¹², polyalkylene glycol    substituted with 0–1 R¹², carbohydrate substituted with 0–1 R¹²,    cyclodextrin substituted with 0–1 R¹², amino acid substituted with    0–1 R¹², polycarboxyalkyl substituted with 0–1 R¹², polyazaalkyl    substituted with 0–1 R¹², peptide substituted with 0–1 R¹², wherein    the peptide is comprised of 2–10 amino acids,    3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and    a bond to C_(h);-   R¹² is a bond to C_(h);-   k is selected from 0, 1, and 2;-   h is selected from 0, 1, and 2;-   h′ is selected from 0, 1, and 2;-   g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   x is selected from 0, 1, 2, 3, 4, and 5;-   x′ is selected from 0, 1, 2, 3, 4, and 5;-   C_(h) is a metal bonding unit having a formula selected from the    group:

-   A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at    each occurrence from the group: NR¹³, NR¹³R¹⁴, S, SH, S(Pg), O, OH,    PR¹³, PR¹³R¹⁴, P(O)R¹⁵R¹⁶, and a bond to L_(n);-   E is a bond, CH, or a spacer group independently selected at each    occurrence from the group: C₁–C₁₀ alkyl substituted with 0–3 R¹⁷,    aryl substituted with 0–3 R¹⁷, C₃₋₁₀ cycloalkyl substituted with 0–3    R¹⁷, heterocyclo-C₁₋₁₀ alkyl substituted with 0–3 R¹⁷, wherein the    heterocyclo group is a 5–10 membered heterocyclic ring system    containing 1–4 heteroatoms independently selected from N, S, and O,    C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0–3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀    aryl substituted with 0–3 R¹⁷, and a 5–10 membered heterocyclic ring    system containing 1–4 heteroatoms independently selected from N, S,    and O and substituted with 0–3 R¹ ⁷;-   R¹³ and R¹⁴ are each independently selected from the group: a bond    to L_(n), hydrogen, C₁–C₁₀ alkyl substituted with 0–3 R¹⁷, aryl    substituted with 0–3 R¹⁷, C₁ ₁₀ cycloalkyl substituted with 0–3 R¹⁷,    heterocyclo-C₁₋₁₀ alkyl substituted with 0–3 R¹⁷, wherein the    heterocyclo group is a 5–10 membered heterocyclic ring system    containing 1–4 heteroatoms independently selected from N, S, and O,    C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0–3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀    aryl substituted with 0–3 R¹⁷, a 5–10 membered heterocyclic ring    system containing 1–4 heteroatoms independently selected from N, S,    and O and substituted with 0–3 R¹⁷, and an electron, provided that    when one of R¹³ or R¹⁴ is an electron, then the other is also an    electron;-   alternatively, R¹³ and R¹⁴ combine to form ═C(R²⁰)(R²¹);-   R¹⁵ and R¹⁶ are each independently selected from the group: a bond    to L_(n), —OH, C₁–C₁₀ alkyl substituted with 0–3 R¹⁷, C₁–C₁₀ alkyl    substituted with 0–3 R¹⁷, aryl substituted with 0–3 R¹⁷, C₃₋₁₀    cycloalkyl substituted with 0–3 R¹⁷, heterocyclo-C₁₋₁₀ alkyl    substituted with 0–3 R¹⁷, wherein the heterocyclo group is a 5–10    membered heterocyclic ring system containing 1–4 heteroatoms    independently selected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl    substituted with 0–3 R¹⁷, C₁₋₁₀ alkyl-C₆₋₁₀ aryl substituted with    0–3 R¹⁷, and a 5–10 membered heterocyclic ring system containing 1–4    heteroatoms independently selected from N, S, and O and substituted    with 0–3 R¹⁷;-   R¹⁷ is independently selected at each occurrence from the group: a    bond to L_(n), ═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R¹⁸, —C(═O)R¹⁸,    —C(═O)N(R¹⁸)₂, —CHO, —CH₂OR¹⁸, —OC(═O)R¹⁸, —OC(═O)OR^(18a), —OR¹⁸,    —OC(═O)N(R¹⁸)₂, —NR¹⁹C(═O)R¹⁸, —NR¹⁹C(═O)OR^(18a),    —NR¹⁹C(═O)N(R¹⁸)₂, —NR¹⁹SO₂N(R¹⁸)₂, —NR¹⁹SO₂R^(18a), —SO₃H,    —SO₂R^(18a), —SR¹⁸, —S(═O)R^(18a), —SO₂N(R¹⁸)₂, —N(R¹⁸)₂,    —NHC(═S)NHR¹⁸, ═NOR¹⁸, NO₂, —C(═O)NHOR¹⁸, —C(═O)NHNR¹⁸R^(18a),    —OCH₂CO₂H, 2-(1-morpholino)ethoxy, C₁–C₅ alkyl, C₂–C₄ alkenyl, C₃–C₆    cycloalkyl, C₃–C₆ cycloalkylmethyl, C₂–C₆ alkoxyalkyl, aryl    substituted with 0–2 R¹⁸, and a 5–10 membered heterocyclic ring    system containing 1–4 heteroatoms independently selected from N, S,    and O;-   R¹⁸, R^(18a), and R¹⁹ are independently selected at each occurrence    from the group: a bond to L_(n), H, C₁–C₆ alkyl, phenyl, benzyl,    C₁–C₆ alkoxy, halide, nitro, cyano, and trifluoromethyl;-   Pg is a thiol protecting group;-   R²⁰ and R²¹ are independently selected from the group: H, C₁–C₁₀    alkyl, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, C₂–C₁₀ 1-alkene    substituted with 0–3 R²³, C₂–C₁₀ 1-alkyne substituted with 0–3 R²³,    aryl substituted with 0–3 R²³, unsaturated 5–10 membered    heterocyclic ring system containing 1–4 heteroatoms independently    selected from N, S, and O and substituted with 0–3 R²³, and    unsaturated C₃₋₁₀ carbocycle substituted with 0–3 R²³;-   alternatively, R²⁰ and R²¹, taken together with the divalent carbon    radical to which they are attached form:

-   R²² and R²³ are independently selected from the group: H, R²⁴,    C₁–C₁₀ alkyl substituted with 0–3 R²⁴, C₂–C₁₀ alkenyl substituted    with 0–3 R²⁴, C₂–C₁₀ alkynyl substituted with 0–3 R²⁴, aryl    substituted with 0–3 R²⁴, a 5–10 membered heterocyclic ring system    containing 1–4 heteroatoms independently selected from N, S, and O    and substituted with 0–3 R²⁴, and C₃₋₁₀ carbocycle substituted with    0–3 R²⁴;-   alternatively, R²², R²³ taken together form a fused aromatic or a    5–10 membered heterocyclic ring system containing 1–4 heteroatoms    independently selected from N, S, and O;-   a and b indicate the positions of optional double bonds and n is 0    or 1;-   R²⁴ is independently selected at each occurrence from the group: ═O,    F, Cl, Br, I, —CF₃, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, —N(R²⁵)₃    ⁺, —CH₂OR²⁵, —OC(═O)R²⁵, —OC(═O)OR^(25a), —OR²⁵, —OC(═O)N(R²⁵)₂,    —NR²⁶C(═O)R²⁵, —NR²⁶C(═O)OR^(25a), —NR²⁶C(═O)N(R²⁵)₂,    —NR²⁶SO₂N(R²⁵)₂, —NR²⁶SO₂R^(25a), —SO₃H, —SO₂R^(25a), —SR²⁵,    —S(═O)R^(25a), —SO₂N(R²⁵)₂, —N(R²⁵)₂, —NOR²⁵, —C(═O)NHOR²⁵,    —OCH₂CO₂H, and 2-(1-morpholino)ethoxy; and,-   R²⁵, R^(25a), and R²⁶ are each independently selected at each    occurrence from the group: hydrogen and C₁–C₆ alkyl;    and a pharmaceutically acceptable salt thereof.-   [3] In a more preferred embodiment, the present invention provides a    compound wherein:-   d is selected from 1, 2, 3, 4, and 5;-   d′ is 1–50;-   W is independently selected at each occurrence from the group: O,    NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)NR⁸, C(═O), C(═O)O, OC(═O),    NHC(═S)NH, NHC(═O)NH, SO₂, (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′),    (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0–1 R¹⁰, C₃₋₁₀    cycloalkyl substituted with 0–1 R¹⁰, and a 5–10 membered    heterocyclic ring system containing 1–4 heteroatoms independently    selected from N, S, and O and substituted with 0–1 R¹⁰;-   R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at each    occurrence from the group: H, ═O, COOH, SO₃H, C₁–C₅ alkyl    substituted with 0–1 R¹⁰, aryl substituted with 0–1 R¹⁰, benzyl    substituted with 0–1 R¹⁰, and C₁–C₅ alkoxy substituted with 0–1 R¹⁰,    NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to    C_(h);-   k is 0 or 1;-   s is selected from 0, 1, 2, 3, 4, and 5;-   s′ is selected from 0, 1, 2, 3, 4, and 5;-   s″ is selected from 0, 1, 2, 3, 4, and 5;-   t is selected from 0, 1, 2, 3, 4, and 5;-   A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at    each occurrence from the group: NR¹³, NR¹³R¹⁴, S, SH, S (Pg), OH,    and a bond to L_(n);-   E is a bond, CH, or a spacer group independently selected at each    occurrence from the group: C₁–C₁₀ alkyl substituted with 0–3 R¹⁷,    aryl substituted with 0–3 R¹⁷, C₃₋₁₀ cycloalkyl substituted with 0–3    R¹⁷, and a 5–10 membered heterocyclic ring system containing 1–4    heteroatoms independently selected from N, S, and O and substituted    with 0–3 R¹⁷;-   R¹³, and R¹⁴ are each independently selected from the group: a bond    to L_(n), hydrogen, C₁–C₁₀ alkyl substituted with 0–3 R¹⁷, aryl    substituted with 0–3 R¹⁷, a 5–10 membered heterocyclic ring system    containing 1–4 heteroatoms independently selected from N, S, and O    and substituted with 0–3 R¹⁷, and an electron, provided that when    one of R¹³ or R¹⁴ is an electron, then the other is also an    electron;-   alternatively, R¹³ and R¹⁴ combine to form ═C(R²⁰)(R²¹);-   R¹⁷ is independently selected at each occurrence from the group: a    bond to L_(n), ═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R¹⁸, —C(═O)R¹⁸,    —C(═O)N(R¹⁸)₂, —CH₂OR¹⁸, —OC(═O)R¹⁸, —OC(═O)OR^(18a), —OR¹⁸,    —OC(═O)N(R¹⁸)₂, —NR¹⁹C(═O)R¹⁸, NR¹⁹C(═O)OR^(18a), —NR¹⁹C(═O)N(R¹⁸)₂,    —NR¹⁹SO₂N(R¹⁸)₂, —NR¹⁹SO₂R^(18a), —SO₃H, —SO₂R^(18a), —S(═O)R^(18a),    —SO₂N(R¹⁸)₂, —N(R¹⁸)₂, —NHC(═S)NHR¹⁸, ═NOR¹⁸, —C(═O)NHNR¹⁸R^(18a),    —OCH₂CO₂H, and 2-(1-morpholino)ethoxy;-   R¹⁸, R^(18a), and R¹⁹ are independently selected at each occurrence    from the group: a bond to L_(n), H, and C₁–C₆ alkyl;-   R²⁰ and R²¹ are independently selected from the group: H, C₁–C₅    alkyl, —CO₂R²⁵, C₂–C₅ 1-alkene substituted with 0–3 R²³, C₂–C₅    1-alkyne substituted with 0–3 R²³, aryl substituted with 0–3 R²³,    and unsaturated 5–10 membered heterocyclic ring system containing    1–4 heteroatoms independently selected from N, S, and O and    substituted with 0–3 R²³;-   alternatively, R²⁰ and R²¹, taken together with the divalent carbon    radical to which they are attached form:

-   R²² and R²³ are independently selected from the group: H, and R²⁴;-   alternatively, R²², R²³ taken together form a fused aromatic or a    5–10 membered heterocyclic ring system containing 1–4 heteroatoms    independently selected from N, S, and O;-   R²⁴ is independently selected at each occurrence from the group:    —CO₂R²⁵, —C(═O)N(R²⁵)₂, —CH₂OR²⁵, —OC(═O)R²⁵, —OR²⁵, —SO₃H,    —N(R²⁵)₂, and —OCH₂CO₂H; and,-   R²⁵ is independently selected at each occurrence from the group: H    and C₁–C₃ alkyl.-   [4] In an even more preferred embodiment, the present invention    provides a compound wherein:-   R^(4a) is benzyl substituted with a bond to L_(n);-   R^(2b) is

-   C_(h) is

-   A¹ is selected from the group: OH, and a bond to L_(n);-   A², A⁴, and A⁶ are each N;-   A³, A⁵, and A⁸ are each OH;-   A⁷ is a bond to L_(n) or NH-bond to L_(n);-   E is a C₂ alkyl substituted with 0–1 R¹⁷;-   R¹⁷ is ═O;-   alternatively, C_(h) is

-   A¹ is selected from the group: OH, and a bond to L_(n);-   A², A³ and A⁴ are each N;-   A⁵, A⁶ and A⁸ are each OH;-   A⁷ is a bond to L_(n);-   E is a C₂ alkyl substituted with 0–1 R¹⁷;-   R¹⁷ is ═O;-   alternatively, C_(h) is

-   A¹ is NH₂ or N═C(R²⁰)(R²¹);-   E is a bond;-   A² is NHR¹³;-   R¹³ is a heterocycle substituted with R¹⁷, the heterocycle being    selected from pyridine and pyrimidine;-   R¹⁷ is selected from a bond to L_(n), C(═O)NHR¹⁸ and C(═O)R¹⁸;-   R¹⁸ is a bond to L_(n);-   R²⁴ is selected from the group: —CO₂R²⁵, —OR²⁵, —SO₃H, and —N(R²⁵)₂;    and,-   R²⁵ is independently selected at each occurrence from the group:    hydrogen and methyl.-   [5] In another even more preferred embodiment, the present invention    provides a compound selected from the group:    -   (S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoic        acid;    -   (S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic        acid;    -   (S)-2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic        acid;    -   (S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic        acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonic        acid;    -   (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)butanoic        acid;    -   (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic        acid;    -   (S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoic        acid;    -   (S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoic        acid;    -   (S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic        acid;    -   (S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)hexanoylamino)butanoic        acid;    -   (S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic        acid;    -   (S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoic        acid;    -   4-[N-(3-{(2R)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl](4S)-4-[(4S)-4-(N-{(1S)-1-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-3-carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}butanoylamino]butanoic        acid;    -   2-(4-{3-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]propyl}(2S)-7-{N-[2-(amidinoamino)ethyl]-N-methylcarbamoyl}-3-oxo-1H,2H,5H-benzo[f]1,4-diazepin-2-yl)acetic        acid; and    -   2-[9-(N-{6-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]hexyl}-N-(benzimidazol-2-ylmethyl)carbamoyl)(5S)-5,6,11-trihydrodibenzo[b,e][7]annulen-5-yl]acetic        acid;        or a pharmaceutically acceptable salt form thereof.-   [6] In a further preferred embodiment, the present invention    provides a kit comprising a compound of the present invention, or a    pharmaceutically acceptable salt form thereof and a pharmaceutically    acceptable carrier.-   [7] In an even further preferred embodiment, the kit further    comprises one or more ancillary ligands and a reducing agent.-   [8] In a still further preferred embodiment, the ancillary ligands    are tricine and TPPTS.-   [9] In another still further preferred embodiment, the reducing    agent is tin(II).-   [10] In a second embodiment, the present invention provides a novel    diagnostic or therapeutic metallopharmaceutical composition,    comprising: a metal, a chelator capable of chelating the metal and a    targeting moiety, wherein the targeting moiety is bound to the    chelator, is a benzodiazepine, benzodiazepinedione, or    dibenzotrihydroannulene nonpeptide and binds to a receptor that is    upregulated during angiogenesis and the compound has 0–1 linking    groups between the targeting moiety and chelator.-   [11] In a preferred embodiment, the metallopharmaceutical is a    diagnostic radiopharmaceutical, the metal is a radioisotope selected    from the group: ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, and ⁶⁸Ga,    and the linking group is present between the targeting moiety and    chelator.-   [12] In another preferred embodiment, the targeting moiety is a    benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and    the receptor is •_(v)•₃ or •_(v)•₅.-   [13] In another preferred embodiment, the radioisotope is ^(99m)Tc    or ⁹⁵Tc, the radiopharmaceutical further comprises a first ancillary    ligand and a second ancillary ligand capable of stabilizing the    radiopharmaceutical.-   [14] In another preferred embodiment, the radioisotope is ^(99m)Tc.-   [15] In another preferred embodiment, the radiopharmaceutical is    selected from the group:-   ^(99m)Tc((S)-2-(2,5-diaza-5-(6((6-(diazenido)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic    acid)(tricine)(TPPTS) and-   ^(99m)Tc((S)-2-(2,5-diaza-9-(N-(6-((6-(diazenido)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic    acid)(tricine)(TPPTS);-   [16] In another preferred embodiment, the radioisotope is ¹¹¹In.-   [17] In another preferred embodiment, the radiopharmaceutical is    selected from the group:-   ¹¹¹In complex of    6-(N-(3-(3-aza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-((2-((carboxymethyl)(2-((carboxymethyl)methylamino)ethyl)amino)ethyl)(2-((carboxymethyl)ethylamino)ethyl)amino)acetylamino)-4-oxooctane-1,8-dicarboxylic    acid;-   ¹¹¹In complex of    (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)butanoic    acid; and-   ¹¹¹In complex of    (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic    acid.-   [18] In another preferred embodiment wherein the    metallopharmaceutical is a therapeutic radiopharmaceutical, the    metal is a radioisotope selected from the group: ³³P, ¹²⁵I, ¹⁸⁶Re,    ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd,    ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag,    and ¹⁹²Ir, and the linking group is present between the targeting    moiety and chelator.-   [19] In another preferred embodiment, the targeting moiety is a    benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and    the receptor is α_(v)β₃ or α_(v)β₅.-   [20] In another preferred embodiment, the radioisotope is ¹⁴⁹Pm.-   [21] In another preferred embodiment, the radiopharmaceutical is    selected from the group:    -   the Pm-149 complex of        (S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoic        acid; and    -   the Pm-149 complex of        (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)butanoic        acid.-   [22] In another preferred embodiment, the radioisotope is ¹⁷⁷Lu.-   [23] In another preferred embodiment, the radiopharmaceutical is    selected from the group:    -   the Lu-177 complex of        (S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoic        acid; and    -   the Lu-177 complex of        (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)butanoic        acid; and    -   the Lu-177 complex of        (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic        acid.-   [24] In another preferred embodiment, the radioisotope is ⁹⁰Y.-   [25] In another preferred embodiment, the radiopharmaceutical is    selected from the group:    -   the Y-90 complex of        (S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoic        acid; and    -   the Y-90 complex of        (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)butanoic        acid; and    -   the Y-90 complex of        (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoic        acid.-   [26] In another preferred embodiment wherein the    metallopharmaceutical is a MRI contrast agent, the metal is a    paramagnetic metal ion selected from the group: Gd(III), Dy(III),    Fe(III), and Mn(II), and the linking group is present between the    targeting moiety and chelator.-   [27] In another preferred embodiment, the targeting moiety is a    benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene and    the receptor is α_(v)β₃ or α_(v)β₅.-   [28] In another preferred embodiment, the metal ion is Gd(III).-   [29] In yet another preferred embodiment wherein the    metallopharmaceutical is a X-ray contrast agent, the metal is    selected from the group: Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au,    Au, Yb, Dy, Cu, Rh, Ag, and Ir, and the linking group is present    between the targeting moiety and chelator.-   [30] In another preferred embodiment, the present invention provides    a novel method of treating rheumatoid arthritis in a patient    comprising: administering a therapeutic radiopharmaceutical capable    of localizing in new angiogenic vasculature to a patient by    injection or infusion wherein the radiopharmaceutical is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator, wherein the metallopharmaceutical is atherapeutic radiopharmaceutical, the metal is a radioisotope selectedfrom the group: ³³p, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm,⁹⁰y, ²¹²Bi, ¹⁰³pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb,¹⁶⁵Dy, ⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir, and the linking group ispresent between the targeting moiety and chelator.

-   [31] In another preferred embodiment, the present invention provides    a novel method of treating cancer in a patient comprising:    administering to a patient in need thereof a therapeutic    radiopharmaceutical by injection or infusion wherein the    radiopharmaceutical is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator, wherein the metallopharmaceutical is atherapeutic radiopharmaceutical, the metal is a radioisotope selectedfrom the group: ³³p, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm,⁹⁰Y, ²¹²Bi, ¹⁰³pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb,¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir, and the linking group ispresent between the targeting moiety and chelator.

-   [32] In another preferred embodiment, the present invention provides    a novel method of treating restenosis in a patient comprising:    administering to a patient, either systemically or locally, a    therapeutic radiopharmaceutical capable of localizing in the    restenotic area and delivering an effective dose of radiation    wherein the radiopharmaceutical is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator, wherein the metallopharmaceutical is atherapeutic radiopharmaceutical, the metal is a radioisotope selectedfrom the group: ³³p, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm,⁹⁰Y, ²¹²Bi, ¹⁰³pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb,¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir, and the linking group ispresent between the targeting moiety and chelator.

-   [33] In another preferred embodiment, the present invention provides    a novel method of imaging cancer in a patient comprising: (1)    administering a diagnostic radiopharmaceutical to a patient by    injection or infusion; (2) imaging the patient using planar or SPECT    gamma scintigraphy, or positron emission tomography wherein the    radiopharmaceutical is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator, wherein the metallopharmaceutical is adiagnostic radiopharmaceutical, the metal is a radioisotope selectedfrom the group: ⁹⁹Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, and ⁶⁸Ga, and thelinking group is present between the targeting moiety and chelator.

-   [34] In another preferred embodiment, the present invention provides    a novel method of imaging cancer in a patient comprising: (1)    administering a MRI contrast agent; and (2) imaging the patient    using magnetic resonance imaging wherein the contrast agent is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator wherein the metallopharmaceutical is a MRIcontrast agent, the metal is a paramagnetic metal ion selected from thegroup: Gd(III),Dy(III), Fe(III), and Mn(II), and the linking group ispresent between the targeting moiety and chelator.

-   [35] In another preferred embodiment, the present invention provides    a novel method of imaging cancer in a patient comprising: (1)    administering an X-ray contrast agent; and (2) imaging the patient    using X-ray computed tomography wherein the contrast agent is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator, wherein the metallopharmaceutical is aX-ray contrast agent, the the metal is selected from the group: Re, Sm,Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy, Cu, Rh, Ag, and Ir, andthe linking group is present between the targeting moiety and chelator.

-   [36] In another preferred embodiment, the present invention provides    a novel method of imaging therapeutic angiogenesis in a patient    comprising: (1) administering a diagnostic radiopharmaceutical, a    MRI contrast agent, or a X-ray contrast agent to a patient by    injection or infusion; (2) imaging the area of the patient wherein    the desired formation of new blood vessels is located wherein the    radiopharmaceutical or contrast agent is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator.

-   [37] In another preferred embodiment, the present invention provides    a novel method of imaging atherosclerosis in a patient    comprising: (1) administering a diagnostic radiopharmaceutical, a    MRI contrast agent, or a X-ray contrast agent of Claim 10 to a    patient by injection or infusion; (2) imaging the area of the    patient wherein the atherosclerosis is located wherein the    radiopharmaceutical or contrast agent is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator.

-   [38] In another even more preferred embodiment, the present    invention provides a novel method of imaging restenosis in a patient    comprising: (1) administering a diagnostic radiopharmaceutical, a    MRI contrast agent, or a X-ray contrast agent to a patient by    injection or infusion; (2) imaging the area of the patient wherein    the restenosis is located wherein the radiopharmaceutical or    contrast agent is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator.

-   [39] In another even more preferred embodiment, the present    invention provides a novel method of imaging cardiac ischemia in a    patient comprising: (1) administering a diagnostic    radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent    to a patient by injection or infusion; (2) imaging the area of the    myocardium wherein the ischemic region is located wherein the    radiopharmaceutical or contrast agent is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator.

-   [40] In another even more preferred embodiment, the present    invention provides a novel method of imaging myocardial reperfusion    injury in a patient comprising: (1) administering a diagnostic    radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent    to a patient by injection or infusion; (2) imaging the area of    myocardium wherein the reperfusion injury is located wherein the    radiopharmaceutical or contrast agent is:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator.

-   [41] In a third embodiment, the present invention provides a novel    compound, comprising: a targeting moiety and a surfactant, wherein    the targeting moiety is bound to the surfactant, is a    benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene    nonpeptide, and binds to a receptor that is upregulated during    angiogenesis and the compound has 0–1 linking groups between the    targeting moiety and surfactant.-   [42] In a preferred embodiment, the receptor is the integrin α_(v)β₃    or α_(v)β₅ and the compound is of the formula:    (Q)_(d)-L_(n)-S_(f)    wherein, Q is a compound of Formulae (Ia), (Ib) or (Ic):

wherein:

-   R¹ and R³ are independently selected from the group: C₁–C₆ alkyl,    benzyl, phenethyl, and a bond to L_(n); provided that one of R¹ and    R³ is a bond to L_(n);-   R² is independently selected from the group: 2-benzimidazolylmethyl,    2-guanidinoethyl, 2-amino-2-pyridyl, 2-amino-2-pyridylmethyl,    5-amino-2-imidazolylmethyl, and 2-imidazolylmethyl;-   R⁴ is independently selected from H, C₁₋₆ alkyl or benzyl;-   R^(2a) is (CH₂)₃R^(3a);-   R^(3a) is selected from the group:

-   R^(4a) is independently selected from C₁₋₆ alkyl substituted with a    bond to L_(n) or benzyl substituted with a bond to L_(n);-   R^(2b) is independently selected from the group:

the asterisks * denote optional positions for attaching L_(n);

-   or Q is a peptide selected from the group:

-   R^(1p) is L-valine, D-valine or L-lysine optionally substituted on    the • amino group with a bond to L_(n);-   R^(2p) is L-phenylalanine, D-phenylalanine, D-1-naphthylalanine,    2-aminothiazole-4-acetic acid or tyrosine, the tyrosine optionally    substituted on the hydroxy group with a bond to L_(n);-   R^(3p) is D-valine;-   R^(4p) is D-tyrosine substituted on the hydroxy group with a bond to    L_(n);-   provided that one of R^(1p) and R^(2p) in each Q is substituted with    a bond to L_(n), and further provided that when R^(2p) is    2-aminothiazole-4-acetic acid, K is N-methylarginine;-   provided that at least one Q is a compound of Formula Ia Ib, or Ic;-   d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   L_(n) is a linking group having the formula:    (W)_(h)—(CR⁶R⁷)_(g))_(x)-(Z)_(k)-((CR^(6a)R^(7a))_(g′)—(W)_(h′))_(x′);-   W is independently selected at each occurrence from the group: O, S,    NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)NR⁸, C(═O), C(═O)O, OC(═O),    NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, (OCH₂CH₂)₂₀₋₂₀₀, (CH₂CH₂O)₂₀₋₂₀₀,    (OCH₂CH₂CH₂)₂₀₋₂₀₀, (CH₂CH₂CH₂O)₂₀₋₂₀₀, and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0–3 R¹⁰, C₃₋₁₀    cycloalkyl substituted with 0–3 R¹⁰, and a 5–10 membered    heterocyclic ring system containing 1–4 heteroatoms independently    selected from N, S, and O and substituted with 0–3 R¹⁰;-   R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at each    occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁–C₅ alkyl    substituted with 0–3 R¹⁰, aryl substituted with 0–3 R¹⁰, benzyl    substituted with 0–3 R¹⁰, and C₁–C₅ alkoxy substituted with 0–3 R¹⁰,    NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to    S_(f);-   R¹⁰ is independently selected at each occurrence from the group: a    bond to S_(f), COOR¹¹, C(═O)NHR¹¹, NHC(═O)R¹¹, OH, NHR¹¹, SO₃H,    PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0–3 R¹¹, C₁₋₅ alkyl    substituted with 0–1 R¹², C₁₋₅ alkoxy substituted with 0–1 R¹², and    a 5–10 membered heterocyclic ring system containing 1–4 heteroatoms    independently selected from N, S, and O and substituted with 0–3    R¹¹;-   R¹¹ is independently selected at each occurrence from the group: H,    alkyl substituted with 0–1 R¹², aryl substituted with 0–1 R¹², a    5–10 membered heterocyclic ring system containing 1–4 heteroatoms    independently selected from N, S, and O and substituted with 0–1    R¹², C₃₋₁₀ cycloalkyl substituted with 0–1 R¹², and a bond to S_(f);-   R¹² is a bond to S_(f);-   k is selected from 0, 1, and 2;-   h is selected from 0, 1, and 2;-   h′ is selected from 0, 1, and 2;-   g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   x is selected from 0, 1, 2, 3, 4, and 5;-   x′ is selected from 0, 1, 2, 3, 4, and 5;-   S_(f) is a surfactant which is a lipid or a compound of the formula:

-   A⁹ is selected from the group: OH and OR²⁷;-   A¹⁰ is OR²⁷;-   R²⁷ is C(═O)C₁₋₂₀ alkyl;-   E¹ is C₁₋₁₀ alkylene substituted with 1–3 R²⁸;-   R²⁸ is independently selected at each occurrence from the group:    R³⁰, —PO₃H—R³⁰, ═O, —CO₂R²⁹, —C(═O)R²⁹, —C(═O)N(R²⁹)₂, —CH₂OR²⁹,    —OR²⁹, —N(R²⁹)₂, C₁–C₅ alkyl, and C₂–C₄ alkenyl;-   R²⁹ is independently selected at each occurrence from the group:    R³⁰, H, C₁–C₆ alkyl, phenyl, benzyl, and trifluoromethyl;-   R³⁰ is a bond to L_(n);    and a pharmaceutically acceptable salt thereof.-   [43] In another preferred embodiment, the compound is of the    formula:    Q-L_(n)-S_(f)    wherein: Q is a compound of Formulae (Ia), (Ib), or (Ic):

-   R^(4a) is benzyl substituted with a bond to L_(n);-   R^(2b) is

-   Z is selected from the group: aryl substituted with 0–1 R¹⁰, C₃₋₁₀    cycloalkyl substituted with 0–1 R¹⁰, and a 5–10 membered    heterocyclic ring system containing 1–4 heteroatoms independently    selected from N, S, and O and substituted with 0–1 R¹⁰;-   R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at each    occurrence from the group: H, ═O, COOH, SO₃H, C₁–C₅ alkyl    substituted with 0–1 R¹⁰, aryl substituted with 0–1 R¹⁰, benzyl    substituted with 0–1 R¹⁰, and C₁–C₅ alkoxy substituted with 0–1 R¹⁰,    NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to    S_(f);-   k is 0 or 1;-   S_(f) is a surfactant which is a lipid or a compound of the formula:

-   A⁹ is OR²⁷;-   A¹⁰ is OR²⁷;-   R²⁷ is C(═O)C₁₋₁₅ alkyl;-   E¹ is C₁₋₄ alkylene substituted with 1–3 R²⁸;-   R²⁸ is independently selected at each occurrence from the group:    R³⁰, —PO₃H—R³⁰, ═O, —CO₂R²⁹, —C(═O)R²⁹, —CH₂OR²⁹, —OR²⁹, and C₁–C₅    alkyl;-   R²⁹ is independently selected at each occurrence from the group:    R³⁰, H, C₁–C₆ alkyl, phenyl, and benzyl;-   R³⁰ is a bond to L_(n);    and a pharmaceutically acceptable salt thereof.-   [44] In another preferred embodiment, the compound selected from the    group:-   Sodium    1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic    acid-dodecoanoate conjugate;-   DPPE-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic    acid]-dodecoanoate conjugate; and-   [(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic    acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)carbamoyl)propyl)carbamoyl]-w-amino-PEG₃₄₀₀-dodecanoate-DPPE    conjugate.-   [45] In another more preferred embodiment, the present invention    provides a novel ultrasound contrast agent composition, comprising:

(a) a compound of comprising: a target moiety an a surfactant, whereinthe target moiety is bount to the surfactant, is a benzodiazepine,benzodiazepinedione, or dibenzotrihydroannulene that binds to theintegrin α_(v)β₃, or α_(v)β₅ and is unregulated during angiogenesis, asurfactant and a linking group between the benzodiazepine and thesurfactant;

(b) a parenterally acceptable carrier; and,

(c) an echogenic gas.

-   [46] In another preferred embodiment, the present invention provides    a novel ultrasound contrast agent composition, further comprising:    1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,    1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, and    N-(methoxypolyethylene glycol 5000    carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.-   [47] In another preferred embodiment, the echogenic gas is a C₂₋₅    perfluorocarbon.-   [48] In another preferred embodiment, the present invention provides    a method of imaging cancer in a patient comprising: (1)    administering, by injection or infusion, a ultrasound contrast agent    composition to a patient; and (2) imaging the patient using    sonography wherein the contrast agent conposition is:

An ultrasound contrast agent composition, comprising:

-   -   (a) a compound comprising: a targeting moiety and a surfactant,        wherein the targeting moiety is bound to the surfactant, is a        benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene        nonpeptide that binds to the integrin α_(v)β_(3,) or α_(v)β₅, a        surfactant and a linking group between the targeting moiety and        surfactant;    -   (b) a parenterally acceptable carrier; and,    -   (c) an echogenic gas.

-   [49] In another preferred embodiment, the present invention provides    a method of imaging therapeutic angiogenesis in a patient    comprising: (1) administering, by injection or infusion, an    ultrasound contrast agent composition to a patient; (2) imaging the    area of the patient wherein the desired formation of new blood    vessels is located wherein the contrast agent composition is:

An ultrasound contrast agent composition, comprising:

-   -   (a) a compound comprising: a targeting moiety and a surfactant,        wherein the targeting moiety is bound to the surfactant, is a        benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene        nonpeptide that binds to the integrin α_(v)β_(3,) or α_(v)β₅, a        surfactant and a linking group between the targeting moiety and        surfactant;    -   (b) a parenterally acceptable carrier; and,    -   (c) an echogenic gas.

-   [50] In another preferred embodiment, the present invention provides    a method of imaging atherosclerosis in a patient comprising: (1)    administering, by injection or infusion, an ultrasound contrast    agent composition of to a patient; (2) imaging the area of the    patient wherein the atherosclerosis is located wherein the contrast    agent composition is:

An ultrasound contrast agent composition, comprising:

-   -   (a) a compound comprising: a targeting moiety and a surfactant,        wherein the targeting moiety is bound to the surfactant, is a        benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene        nonpeptide that binds to the integrin α_(v)β_(3,) or α_(v)β₅, a        surfactant and a linking group between the targeting moiety and        surfactant;    -   (b) a parenterally acceptable cazrrier; and,    -   (c) an echogenic gas.

-   [51] In another preferred embodiment, the present invention provides    a method of imaging restenosis in a patient comprising: (1)    administering, by injection or infusion, an ultrasound contrast    agent composition of to a patient; (2) imaging the area of the to    patient wherein the restenosis is located wherein the contrast agent    composition is:

An ultrasound contrast agent composition, comprising:

-   -   (a) a compound comprising: a targeting moiety and a surfactant,        wherein the targeting moiety is bound to the surfactant, is a        benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene        nonpeptide that binds to the integrin α_(v)β_(3,) or α_(v)β₅, a        surfactant and a linking group between the targeting moiety and        surfactant;    -   (b) a parenterally acceptable cazrrier; and,    -   (c) an echogenic gas.

-   [52] In another preferred embodiment, the present invention provides    a method of imaging cardiac ischemia in a patient comprising: (1)    administering, by injection or infusion, an ultrasound contrast    agent composition of to a patient; (2) imaging the area of the    myocardium wherein the ischemic region is located wherein the    contrast agent composition is:

An ultrasound contrast agent composition, comprising:

-   -   (a) a compound comprising: a targeting moiety and a surfactant,        wherein the targeting moiety is bound to the surfactant, is a        benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene        nonpeptide that binds to the integrin α_(v)β_(3,) or α_(v)β₅, a        surfactant and a linking group between the targeting moiety and        surfactant;    -   (b) a parenterally acceptable cazrrier; and,    -   (c) an echogenic gas.

-   [53] In another preferred embodiment, the present invention provides    a method of imaging myocardial reperfusion injury in a patient    comprising: (1) administering, by injection or infusion, an    ultrasound contrast agent composition to a patient; (2) imaging the    area of myocardium wherein the reperfusion injury is located wherein    the contrast agent composition is:

An ultrasound contrast agent composition, comprising:

-   -   (a) a compound comprising: a targeting moiety and a surfactant,        wherein the targeting moiety is bound to the surfactant, is a        benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulene        nonpeptide that binds to the integrin α_(v)β_(3,) or α_(v)β₅, a        surfactant and a linking group between the targeting moiety and        surfactant;    -   (b) a parenterally acceptable cazrrier; and,    -   (c) an echogenic gas.

-   [54] In another preferred embodiment, the present invention provides    a novel therapeutic radiopharmaceutical composition, comprising:

(a) a therapeutic radiopharmaceutical and,

(b) a parenterally acceptable carrier wherein the radiopharmaceuticalis:

A diagnostic or therapeutic metallopharmaceutical composition,comprising: a metal, a chelator capable of chelating the metal and atargeting moiety, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide and binds to a receptor that is upregulated duringangiogenesis and the compound has 0-1 linking groups between thetargeting moiety and chelator, wherein the metallopharmaceutical is atherapeutic radiopharmaceutical, the metal is a radioisotope selectedfrom the group: ³³p, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm,⁹⁰Y, ²¹²Bi, ¹⁰³pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb,¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir, and the linking group ispresent between the targeting moiety and chelator; and the receptor isα_(v)β₃ or α_(v)β₅.

-   [55] In another preferred embodiment, the present invention provides    a novel diagnostic radiopharmaceutical composition, comprising:

(a) a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-raycontrast agent of Claim 10; and,

(b) a parenterally acceptable carrier.

-   [56] In another preferred embodiment, the present invention provides    a kit for treating cancer, comprising a compound of Embodiment 1, or    a pharmaceutically acceptable salt thereof, and at least one agent    selected from the group consisting of a chemotherapeutic agent and a    radiosensitizer agent, or a pharmaceutically acceptable salt    thereof, and a pharmaceutically acceptable carrier.-   [57] In another preferred embodiment, the present invention provides    a kit according to Embodiment 58 wherein said kit comprises a    plurality of separate containers, wherein at least one of said    containers contains a compound of Embodiment 1, or a    pharmaceutically acceptable salt thereof, and at least another of    said containers contains one or more agents selected from the group    consisting of a chemotherapeutic agent and a radiosensitizer agent,    or a pharmaceutically acceptable salt thereof, and a    pharmaceutically acceptable carrier.-   [58] In another preferred embodiment, the present invention provides    a kit according to Embodiment 56, wherein the chemotherapeutic agent    is selected from the group consisting of mitomycin, tretinoin,    ribomustin, gemcitabine, vincristine, etoposide, cladribine,    mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin,    nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed,    daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane,    nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone,    aminoglutethimide, amsacrine, proglumide, elliptinium acetate,    ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin,    nimustine, vindesine, flutamide, drogenil, butocin, carmofur,    razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,    prednimustine, picibanil, levamisole, teniposide, improsulfan,    enocitabine, lisuride, oxymetholone, tamoxifen, progesterone,    mepitiostane, epitiostanol, formestane, interferon-alpha,    interferon-2 alpha, interferon-beta, interferon-gamma, colony    stimulating factor-1, colony stimulating factor-2, denileukin    diftitox, interleukin-2, and leutinizing hormone releasing factor.-   [59] In another preferred embodiment, the present invention provides    a kit according to Embodiment 56, wherein the chemotherapeutic agent    is selected from the group consisting of mitomycin, tretinoin,    ribomustin, gemcitabine, vincristine, etoposide, cladribine,    mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin,    nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed,    daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane,    nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone,    aminoglutethimide, amsacrine, proglumide, elliptinium acetate,    ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin,    nimustine, vindesine, flutamide, drogenil, butocin, carmofur,    razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,    prednimustine, picibanil, levamisole, teniposide, improsulfan,    enocitabine, and lisuride.-   [60] In another preferred embodiment, the present invention provides    a kit according to Embodiment 56 wherein the chemotherapeutic agent    is selected from the group consisting of oxymetholone, tamoxifen,    progesterone, mepitiostane, epitiostanol, and formestane.-   [61] In another preferred embodiment, the present invention provides    a kit according to Embodiment 56 wherein the chemotherapeutic agent    is selected from the group consisting of interferon-alpha,    interferon-2 alpha, interferon-beta, interferon-gamma, colony    stimulating factor-1, colony stimulating factor-2, denileukin    diftitox, interleukin-2, and leutinizing hormone releasing factor.-   [62] In another preferred embodiment, the present invention provides    a kit according to Embodiment 56, wherein radiosensitizer agent is    selected from the group consiting of    2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,    N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,    3-amino-1,2,4-benzotriazine-1,4-dioxide,    N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,    1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and    1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.-   [63] In another preferred embodiment, the present invention provides    a therapeutic metallopharmaceutical composition according to    Embodiment 10, wherein the metallopharmaceutical is a therapeutic    radiopharmaceutical, further comprising at least one agent selected    from the group consisting of a chemotherapeutic agent and a    radiosensitizer agent, or a pharmaceutically acceptable salt    thereof.-   [64] In another preferred embodiment, the present invention provides    a therapeutic metallopharmaceutical composition according to    Embodiment 63, wherein the chemotherapeutic agent is selected from    the group consisting of mitomycin, tretinoin, ribomustin,    gemcitabine, vincristine, etoposide, cladribine, mitobronitol,    methotrexate, doxorubicin, carboquone, pentostatin, nitracrine,    zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin,    fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin,    cytarabine, bicalutamide, vinorelbine, vesnarinone,    aminoglutethimide, amsacrine, proglumide, elliptinium acetate,    ketanserin, doxifluridine, etretinate, isotretitoin, streptozocin,    nimustine, vindesine, flutamide, drogenil, butocin, carmofur,    razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,    prednimustine, picibanil, levamisole, teniposide, improsulfan,    enocitabine, lisuride, oxymetholone, tamoxifen, progesterone,    mepitiostane, epitiostanol, formestane, interferon-alpha,    interferon-2 alpha, interferon-beta, interferon-gamma, colony    stimulating factor-1, colony stimulating factor-2, denileukin    diftitox, interleukin-2, and leutinizing hormone releasing factor.-   [65] In another preferred embodiment, the present invention provides    a therapeutic metallopharmaceutical composition according to    Embodiment 63, wherein radiosensitizer agent is selected from the    group consiting of    2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,    N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,    3-amino-1,2,4-benzotriazine-1,4-dioxide,    N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,    1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and    1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.-   [66] In another preferred embodiment, the present invention provides    a method of treating cancer in a patient comprising: administering    to a patient in need thereof a metallopharmaceutical of Embodiment    18 or a pharmaceutically acceptable salt thereof, and at least one    agent selected from the group consisting of a chemotherapeutic agent    and a radiosensitizer agent, or a pharmaceutically acceptable salt    thereof.-   [67] In another preferred embodiment, the present invention provides    a method of treating cancer according to Embodiment 66, wherein the    administration is by injection or infusion.-   [68] In another preferred embodiment, the present invention provides    a method according to Embodiment 66 wherein administering the    therapeutic radiopharmaceutical and agent is concurrent.-   [69] In another preferred embodiment, the present invention provides    a method according to Embodiment 66 wherein administering the    therapeutic radiopharmaceutical and agent is sequential.-   [70] In another preferred embodiment, the present invention provides    a method according to Embodiment 66 wherein the cancer is selected    from the group consisting of carcinomas of the lung, breast, ovary,    stomach, pancreas, larynx, esophagus, testes, liver, parotid,    biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,    bladder, prostate,thyroid, squamous cell carcinomas,    adenocarcinomas, small cell carcinomas, melanomas, gliomas, and    neuroblastomas.-   [71] In another preferred embodiment, the present invention provides    a method according to Embodiment 66 wherein the chemotherapeutic    agent is selected from the group consisting of mitomycin, tretinoin,    ribomustin, gemcitabine, vincristine, etoposide, cladribine,    mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin,    nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed,    daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane,    nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone,    aminoglutethimide, amsacrine, proglumide, elliptinium acetate,    ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin,    nimustine, vindesine, flutamide, drogenil, butocin, carmofur,    razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide,    prednimustine, picibanil, levamisole, teniposide, improsulfan,    enocitabine, lisuride, oxymetholone, tamoxifen, progesterone,    mepitiostane, epitiostanol, formestane, interferon-alpha,    interferon-2 alpha, interferon-beta, interferon-gamma, colony    stimulating factor-1, colony stimulating factor-2, denileukin    diftitox, interleukin-2, and leutinizing hormone releasing factor.-   [72] In another preferred embodiment, the present invention provides    a method according to Embodiment 66 wherein the radiosensitizer    agent is selected from the group consiting of    2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,    N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,    3-amino-1,2,4-benzotriazine-1,4-dioxide,    N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,    1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and    1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.-   [73] In another preferred embodiment, the present invention provides    a process for the preparation of diagnostic or therapeutic    metallopharmaceutical composition, said process comprising    generating a macrostructure from a plurality of molecular components    wherein the plurality of components includes a targeting moiety and    a chelator, wherein the targeting moiety is a benzodiazepine,    benzodiazepinedione, or dibenzotrihydroannulene nonpeptide, which is    bound to the chelator, and binds to a receptor that is upregulated    during angiogenesis and the compound has 0–1 linking groups between    the targeting moiety and chelator.

Another aspect of the present invention are diagnostic kits for thepreparation of radiopharmaceuticals useful as imaging agents for cancer.Diagnostic kits of the present invention comprise one or more vialscontaining the sterile, non-pyrogenic, formulation comprised of apredetermined amount of a reagent of the present invention, andoptionally other components such as one or two ancillary ligands,reducing agents, transfer ligands, buffers, lyophilization aids,stabilization aids, solubilization aids and bacteriostats. The inclusionof one or more optional components in the formulation will frequentlyimprove the ease of synthesis of the radiopharmaceutical by thepracticing end user, the ease of manufacturing the kit, the shelf-lifeof the kit, or the stability and shelf-life of the radiopharmaceutical.The inclusion of one or two ancillary ligands is required for diagnostickits comprising reagent comprising a hydrazine or hydrazone bondingmoiety. The one or more vials that contain all or part of theformulation can independently be in the form of a sterile solution or alyophilized solid.

Another aspect of the present invention contemplates a method of imagingcancer in a patient involving: (1) synthesizing a diagnosticradiopharmaceutical of the present invention, using a reagent of thepresent invention, capable of localizing in tumors; (2) administeringsaid radiopharmaceutical to a patient by injection or infusion; (3)imaging the patient using planar or SPECT gamma scintigraphy, orpositron emission tomography.

Another aspect of the present invention contemplates a method of imagingcancer in a patient involving: (1) administering a paramagneticmetallopharmaceutical of the present invention capable of localizing intumors to a patient by injection or infusion; and (2) imaging thepatient using magnetic resonance imaging.

Another aspect of the present invention contemplates a method of imagingcancer in a patient involving: (1) administering a X-ray contrast agentof the present invention capable of localizing in tumors to a patient byinjection or infusion; and (2) imaging the patient using X-ray computedtomography.

Another aspect of the present invention contemplates a method of imagingcancer in a patient involving: (1) administering a ultrasound contrastagent of the present invention capable of localizing in tumors to apatient by injection or infusion; and (2) imaging the patient usingsonography.

Another aspect of the present invention contemplates a method oftreating cancer in a patient involving: (1) administering a therapeuticradiopharmaceutical of the present invention capable of localizing intumors to a patient by injection or infusion.

Another aspect of the present invention contemplates the combination ofchemotherapeutics and angiogenesis-targeted therapeuticradiopharmaceuticals of the invention, which target the luminal side ofthe neovasculature of tumors, to provide a surprising, and enhanceddegree of tumor suppression relative to each treatment modality alonewithout significant additive toxicity.

Another aspect of the present invention contemplates the compounds ofthe present invention (i.e. a compound comprising: a targeting moietyand a chelator, wherein the targeting moiety is bound to the chelator,is a benzodiazepine, benzodiazepinedione, or dibenzotrihydroannulenenonpeptide, and binds to a receptor that is upregulated duringangiogenesis and the compound has 0–1 linking groups between thetargeting moiety and chelator) which is administered in combinationtherapy, with one or more chemotherapeutic agent(s)selected from thegroup consisting of mitomycin, tretinoin, ribomustin, gemcitabine,vincristine, etoposide, cladribine, mitobronitol, methotrexate,doxorubicin, carboquone, pentostatin, nitracrine, zinostatin,cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole,fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine,bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine,proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate,isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil,butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol,tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide,improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen,progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha,interferon-2 alpha, interferon-beta, interferon-gamma, colonystimulating factor-1, colony stimulating factor-2, denileukin diftitox,interleukin-2, and leutinizing hormone releasing factor.

This combination therapy may further, optionally, include aradiosensitizer agent, or a pharmaceutically acceptable salt thereof, toenhance the radiotherapeutic effect together with the chemotherapeuticagent, said radiosensitizer agent being selected from the groupconsisting of2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,3-amino-1,2,4-benzotriazine-1,4-dioxide,N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol. A thoroughdiscussion of radiosensitizer agents is provided in the following:Rowinsky-EK, Oncology-Huntingt., 1999 October; 13(10 Suppl 5): 61–70;Chen-AY et al., Oncology-Huntingt. 1999 October; 13(10 Suppl 5): 39–46;Choy-H, Oncology-Huntingt. 1999 October; 13(10 Suppl 5): 23–38; andHerscher-LL et al, Oncology-Huntingt. 1999 October; 13(10 Suppl 5):11–22, which are incorporated herein by reference.

It is a further aspect of the invention to provide kits having aplurality of active ingredients (with or without carrier) which,together, may be effectively utilized for carrying out the novelcombination therapies of the invention.

It is another aspect of the invention to provide a novel pharmaceuticalcomposition which is effective, in and of itself, for utilization in abeneficial combination therapy because it includes compounds of thepresent invention, and a chemotherapeutic agent or a radiosensitizeragent, which may be utilized in accordance with the invention.

In another aspect, the present invention provides a method for treatingcancer in a patient in need of such treatment, said method including thesteps of administering a therapeutically effective amount of a compoundof the present invention and administering a therapeutically effectiveamount of at least one agent selected from the group consisting of achemotherapeutic agent and a radiosensitizer agent.

It is to be understood that this invention covers all appropriatecombinations of the particular and preferred groupings and embodimentsreferred to herein.

DEFINITIONS

The compounds herein described may have asymmetric centers. Unlessotherwise indicated, all chiral, diastereomeric and racemic forms areincluded in the present invention. Many geometric isomers of olefins,C═N double bonds, and the like can also be present in the compoundsdescribed herein, and all such stable isomers are contemplated in thepresent invention. It will be appreciated that compounds of the presentinvention contain asymmetrically substituted carbon atoms, and may beisolated in optically active or racemic forms. It is well known in theart how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Two distinct isomers (cis and trans) of the peptide bond are known tooccur; both can also be present in the compounds described herein, andall such stable isomers are contemplated in the present invention. The Dand L-isomers of a particular amino acid are designated herein using theconventional 3-letter abbreviation of the amino acid, as indicated bythe following examples: D-Leu, or L-Leu.

When any variable occurs more than one time in any substituent or in anyformula, its definition on each occurrence is independent of itsdefinition at every other occurrence. Thus, for example, if a group isshown to be substituted with 0–2 R⁵², then said group may optionally besubstituted with up to two R⁵², and R⁵² at each occurrence is selectedindependently from the defined list of possible R⁵². Also, by way ofexample, for the group —N(R⁵³)₂, each of the two R⁵³ substituents on Nis independently selected from the defined list of possible R⁵³.Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds. When a bond to asubstituent is shown to cross the bond connecting two atoms in a ring,then such substituent may be bonded to any atom on the ring.

The term “nonpeptide” means preferably less than three amide bonds inthe backbone core of the targeting moiety or preferably less than threeamino acids or amino acid mimetics in the targeting moiety.

The term “metallopharmaceutical” means a pharmaceutical comprising ametal. The metal is the cause of the imageable signal in diagnosticapplications and the source of the cytotoxic radiation inradiotherapeutic applications. Radiopharmaceuticals aremetallopharmaceuticals in which the metal is a radioisotope.

By “reagent” is meant a compound of this invention capable of directtransformation into a metallopharmaceutical of this invention. Reagentsmay be utilized directly for the preparation of themetallopharmaceuticals of this invention or may be a component in a kitof this invention.

The term “binding agent” means a metallopharmaceutical of this inventionhaving affinity for and capable of binding to the vitronectin receptor.The binding agents of this invention have Ki<1000 nM.

By “stable compound” or “stable structure” is meant herein a compoundthat is sufficiently robust to survive isolation to a useful degree ofpurity from a reaction mixture, and formulation into an efficaciouspharmaceutical agent.

The term “substituted”, as used herein, means that one or more hydrogenson the designated atom or group is replaced with a selection from theindicated group, provided that the designated atom's or group's normalvalency is not exceeded, and that the substitution results in a stablecompound. When a substituent is keto (i.e., ═O), then 2 hydrogens on theatom are replaced.

The term “bond”, as used herein, means either a single or double bond.

The term “salt”, as used herein, is used as defined in the CRC Handbookof Chemistry and Physics, 65th Edition, CRC Press, Boca Raton, Fla.,1984, as any substance which yields ions, other than hydrogen orhydroxyl ions. As used herein, “pharmaceutically acceptable salts” referto derivatives of the disclosed compounds modified by making acid orbase salts. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as carboxylicacids; and the like.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable prodrugs” as used herein meansthose prodrugs of the compounds useful according to the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” means compoundsthat are rapidly transformed in vivo to yield the parent compound of theabove formula, for example by hydrolysis in blood. Functional groupswhich may be rapidly transformed, by metabolic cleavage, in vivo form aclass of groups reactive with the carboxyl group of the compounds ofthis invention. They include, but are not limited to such groups asalkanoyl (such as acetyl, propionyl, butyryl, and the like),unsubstituted and substituted aroyl (such as benzoyl and substitutedbenzoyl), alkoxycarbonyl (such as ethoxycarbonyl), trialkylsilyl (suchas trimethyl- and triethysilyl), monoesters formed with dicarboxylicacids (such as succinyl), and the like. Because of the ease with whichthe metabolically cleavable groups of the compounds useful according tothis invention are cleaved in vivo, the compounds bearing such groupsact as pro-drugs. The compounds bearing the metabolically cleavablegroups have the advantage that they may exhibit improved bioavailabilityas a result of enhanced solubility and/or rate of absorption conferredupon the parent compound by virtue of the presence of the metabolicallycleavable group. A thorough discussion of prodrugs is provided in thefollowing: Design of Prodrugs, H. Bundgaard, ed., Elsevier, 1985;Methods in Enzymology, K. Widder et al, Ed., Academic Press, 42, p.309–396, 1985; A Textbook of Drug Design and Development,Krogsgaard-Larsen and H. Bundgaard, ed., Chapter 5; “Design andApplications of Prodrugs” p. 113–191, 1991; Advanced Drug DeliveryReviews, H. Bundgard, 8, p. 1–38, 1992; Journal of PharmaceuticalSciences, 77, p. 285, 1988; Chem. Pharm. Bull., N. Nakeya et al, 32, p.692, 1984; Pro-drugs as Novel Delivery Systems, T. Higuchi and V.Stella, Vol. 14 of the A.C.S. Symposium Series, and BioreversibleCarriers in Drug Design, Edward B. Roche, ed., American PharmaceuticalAssociation and Pergamon Press, 1987, which are incorporated herein byreference.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, examples of which include, but are notlimited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl;“cycloalkyl” or “carbocycle” is intended to include saturated andpartially unsaturated ring groups, including mono-, bi- or poly-cyclicring systems, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl and adamantyl; “bicycloalkyl” or “bicyclic” isintended to include saturated bicyclic ring groups such as[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane(decalin), [2.2.2]bicyclooctane, and so forth.

As used herein, the term “alkene” or “alkenyl” is intended to includehydrocarbon chains having the specified number of carbon atoms of eithera straight or branched configuration and one or more unsaturatedcarbon-carbon bonds which may occur in any stable point along the chain,such as ethenyl, propenyl, and the like.

As used herein, the term “alkyne” or “alkynyl” is intended to includehydrocarbon chains having the specified number of carbon atoms of eithera straight or branched configuration and one or more unsaturatedcarbon-carbon triple bonds which may occur in any stable point along thechain, such as propargyl, and the like.

As used herein, “aryl” or “aromatic residue” is intended to mean phenylor naphthyl, which when substituted, the substitution can be at anyposition.

As used herein, the term “heterocycle” or “heterocyclic system” isintended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7-to 10-membered bicyclic heterocyclic ring which is saturated partiallyunsaturated or unsaturated (aromatic), and which consists of carbonatoms and from 1 to 4 heteroatoms independently selected from the groupconsisting of N, O and S and including any bicyclic group in which anyof the above-defined heterocyclic rings is fused to a benzene ring. Thenitrogen and sulfur heteroatoms may optionally be oxidized. Theheterocyclic ring may be attached to its pendant group at any heteroatomor carbon atom which results in a stable structure. The heterocyclicrings described herein may be substituted on carbon or on a nitrogenatom if the resulting compound is stable. If specifically noted, anitrogen in the heterocycle may optionally be quaternized. It ispreferred that when the total number of S and O atoms in the heterocycleexceeds 1, then these heteroatoms are not adjacent to one another. It ispreferred that the total number of S and O atoms in the heterocycle isnot more than 1. As used herein, the term “aromatic heterocyclic system”is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or7- to 10-membered bicyclic heterocyclic aromatic ring which consists ofcarbon atoms and from 1 to 4 heteroatoms independently selected from thegroup consisting of N, O and S. It is preferred that the total number ofS and O atoms in the aromatic heterocycle is not more than 1.

Examples of heterocycles include, but are not limited to, 1H-indazole,2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,carbazolyl, 4aH-carbazolyl, β-carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinylperimidinyl,phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl,pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, xanthenyl. Preferred heterocycles include, but are notlimited to, pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl,benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, or isatinoyl.Also included are fused ring and spiro compounds containing, forexample, the above heterocycles.

As used herein, the term “alkaryl” means an aryl group bearing an alkylgroup of 1–10 carbon atoms; the term “aralkyl” means an alkyl group of1–10 carbon atoms bearing an aryl group; the term “arylalkaryl” means anaryl group bearing an alkyl group of 1–10 carbon atoms bearing an arylgroup; and the term “heterocycloalkyl” means an alkyl group of 1–10carbon atoms bearing a heterocycle.

A “polyalkylene glycol” is a polyethylene glycol, polypropylene glycolor polybutylene glycol having a molecular weight of less than about5000, terminating in either a hydroxy or alkyl ether moiety.

A “carbohydrate” is a polyhydroxy aldehyde, ketone, alcohol or acid, orderivatives thereof, including polymers thereof having polymericlinkages of the acetal type.

A “cyclodextrin” is a cyclic oligosaccharide. Examples of cyclodextrinsinclude, but are not limited to, α-cyclodextrin,hydroxyethyl-α-cyclodextrin, hydroxypropyl-α-cyclodextrin,β-cyclodextrin, hydroxypropyl-β-cyclodextrin,carboxymethyl-β-cyclodextrin, dihydroxypropyl-β-cyclodextrin,hydroxyethyl-β-cyclodextrin, 2,6 di-O-methyl-β-cyclodextrin,sulfated-β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin,dihydroxypropyl-γ-cyclodextrin, hydroxyethyl-γ-cyclodextrin, andsulfated γ-cyclodextrin.

As used herein, the term “polycarboxyalkyl” means an alkyl group havingbetween two and about 100 carbon atoms and a plurality of carboxylsubstituents; and the term “polyazaalkyl” means a linear or branchedalkyl group having between two and about 100 carbon atoms, interruptedby or substituted with a plurality of amine groups.

A “reducing agent” is a compound that reacts with a radionuclide, whichis typically obtained as a relatively unreactive, high oxidation statecompound, to lower its oxidation state by transferring electron(s) tothe radionuclide, thereby making it more reactive. Reducing agentsuseful in the preparation of radiopharmaceuticals and in diagnostic kitsuseful for the preparation of said radiopharmaceuticals include but arenot limited to stannous chloride, stannous fluoride, formamidinesulfinic acid, ascorbic acid, cysteine, phosphines, and cuprous orferrous salts. Other reducing agents are described in Brodack et. al.,PCT Application 94/22496, which is incorporated herein by reference.

A “transfer ligand” is a ligand that forms an intermediate complex witha metal ion that is stable enough to prevent unwanted side-reactions butlabile enough to be converted to a metallopharmaceutical. The formationof the intermediate complex is kinetically favored while the formationof the metallopharmaceutical is thermodynamically favored. Transferligands useful in the preparation of metallopharmaceuticals and indiagnostic kits useful for the preparation of diagnosticradiopharmaceuticals include but are not limited to gluconate,glucoheptonate, mannitol, glucarate,N,N,N′,N′-ethylenediaminetetraacetic acid, pyrophosphate andmethylenediphosphonate. In general, transfer ligands are comprised ofoxygen or nitrogen donor atoms.

The term “donor atom” refers to the atom directly attached to a metal bya chemical bond.

“Ancillary” or “co-ligands” are ligands that are incorporated into aradiopharmaceutical during its synthesis. They serve to complete thecoordination sphere of the radionuclide together with the chelator orradionuclide bonding unit of the reagent. For radiopharmaceuticalscomprised of a binary ligand system, the radionuclide coordinationsphere is composed of one or more chelators or bonding units from one ormore reagents and one or more ancillary or co-ligands, provided thatthere are a total of two types of ligands, chelators or bonding units.For example, a radiopharmaceutical comprised of one chelator or bondingunit from one reagent and two of the same ancillary or co-ligands and aradiopharmaceutical comprised of two chelators or bonding units from oneor two reagents and one ancillary or co-ligand are both considered to becomprised of binary ligand systems. For radiopharmaceuticals comprisedof a ternary ligand system, the radionuclide coordination sphere iscomposed of one or more chelators or bonding units from one or morereagents and one or more of two different types of ancillary orco-ligands, provided that there are a total of three types of ligands,chelators or bonding units. For example, a radiopharmaceutical comprisedof one chelator or bonding unit from one reagent and two differentancillary or co-ligands is considered to be comprised of a ternaryligand system.

Ancillary or co-ligands useful in the preparation ofradiopharmaceuticals and in diagnostic kits useful for the preparationof said radiopharmaceuticals are comprised of one or more oxygen,nitrogen, carbon, sulfur, phosphorus, arsenic, selenium, and telluriumdonor atoms. A ligand can be a transfer ligand in the synthesis of aradiopharmaceutical and also serve as an ancillary or co-ligand inanother radiopharmaceutical. Whether a ligand is termed a transfer orancillary or co-ligand depends on whether the ligand remains in theradionuclide coordination sphere in the radiopharmaceutical, which isdetermined by the coordination chemistry of the radionuclide and thechelator or bonding unit of the reagent or reagents.

A “chelator” or “bonding unit” is the moiety or group on a reagent thatbinds to a metal ion through the formation of chemical bonds with one ormore donor atoms.

The term “binding site” means the site in vivo or in vitro that binds abiologically active molecule.

A “diagnostic kit” or “kit” comprises a collection of components, termedthe formulation, in one or more vials which are used by the practicingend user in a clinical or pharmacy setting to synthesize diagnosticradiopharmaceuticals. The kit provides all the requisite components tosynthesize and use the diagnostic radiopharmaceutical except those thatare commonly available to the practicing end user, such as water orsaline for injection, a solution of the radionuclide, equipment forheating the kit during the synthesis of the radiopharmaceutical, ifrequired, equipment necessary for administering the radiopharmaceuticalto the patient such as syringes and shielding, and imaging equipment.

Therapeutic radiopharmaceuticals, X-ray contrast agent pharmaceuticals,ultrasound contrast agent pharmaceuticals and metallopharmaceuticals formagnetic resonance imaging contrast are provided to the end user intheir final form in a formulation contained typically in one vial, aseither a lyophilized solid or an aqueous solution. The end userreconstitutes the lyophilized with water or saline and withdraws thepatient dose or just withdraws the dose from the aqueous solutionformulation as provided.

A “lyophilization aid” is a component that has favorable physicalproperties for lyophilization, such as the glass transition temperature,and is added to the formulation to improve the physical properties ofthe combination of all the components of the formulation forlyophilization.

A “stabilization aid” is a component that is added to themetallopharmaceutical or to the diagnostic kit either to stabilize themetallopharmaceutical or to prolong the shelf-life of the kit before itmust be used. Stabilization aids can be antioxidants, reducing agents orradical scavengers and can provide improved stability by reactingpreferentially with species that degrade other components or themetallopharmaceutical.

A “solubilization aid” is a component that improves the solubility ofone or more other components in the medium required for the formulation.

A “bacteriostat” is a component that inhibits the growth of bacteria ina formulation either during its storage before use of after a diagnostickit is used to synthesize a radiopharmaceutical.

The following abbreviations are used herein:

Acm acetamidomethyl b-Ala, beta-Ala 3-aminopropionic acid or bAla ATA2-aminothiazole-5-acetic acid or 2-aminothiazole-5-acetyl group Boct-butyloxycarbonyl CBZ, Cbz or Z Carbobenzyloxy Cit citrulline Dap2,3-diaminopropionic acid DCC dicyclohexylcarbodiimide DIEAdiisopropylethylamine DMAP 4-dimethylaminopyridine EOE ethoxyethyl HBTU2-(1H-Benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphatehynic boc-hydrazinonicotinyl group or 2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]- benzenesulfonic acid, NNeArg or MeArga-N-methyl arginine NMeAsp a-N-methyl aspartic acid NMMN-methylmorpholine OcHex O-cyclohexyl OBzl O-benzyl oSu O-succinimidylTBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3- tetramethyluroniumtetrafluoroborate THF tetrahydrofuranyl THP tetrahydropyranyl Tos tosylTr trityl

The following conventional three-letter amino acid abbreviations areused herein; the conventional one-letter amino acid abbreviations areNOT used herein:

Ala = alanine Arg = arginine Asn = asparagine Asp = aspartic acid Cys =cysteine Gln = glutamine Glu = glutamic acid Gly = glycine His =histidine Ile = isoleucine Leu = leucine Lys = lysine Met = methionineNle = norleucine Orn = ornithine Phe = phenylalanine Phg = phenylglycinePro = proline Sar = sarcosine Ser = serine Thr = threonine Trp =tryptophan Tyr = tyrosine Val = valine

As used herein, the term “bubbles”, as used herein, refers to vesicleswhich are generally characterized by the presence of one or moremembranes or walls surrounding an internal void that is filled with agas or precursor thereto. Exemplary bubbles include, for example,liposomes, micelles and the like.

As used herein, the term “lipid” refers to a synthetic ornaturally-occurring amphipathic compound which comprises a hydrophiliccomponent and a hydrophobic component. Lipids include, for example,fatty acids, neutral fats, phosphatides, glycolipids, aliphatic alcholsand waxes, terpenes and steroids.

As used herein, the term “lipid composition” refers to a compositionwhich comprises a lipid compound. Exemplary lipid compositions includesuspensions, emulsions and vesicular compositions.

As used herein, the term “lipid formulation” refers to a compositionwhich comprises a lipid compound and a bioactive agent.

As used herein, the term “vesicle” refers to a spherical entity which ischaracterized by the presence of an internal void. Preferred vesiclesare formulated from lipids, including the various lipids describedherein. In any given vesicle, the lipids may be in the form of amonolayer or bilayer, and the mono- or bilayer lipids may be used toform one of more mono- or bilayers. In the case of more than one mono-or bilayer, the mono- or bilayers are generally concentric. The lipidvesicles described herein include such entities commonly referred to asliposomes, micelles, bubbles, microbubbles, microspheres and the like.Thus, the lipids may be used to form a unilamellar vesicle (comprised ofone monolayer or bilayer), an oligolamellar vesicle (comprised of abouttwo or about three monolayers or bilayers) or a multilamellar vesicle(comprised of more than about three monolayers or bilayers). Theinternal void of the vesicles may be filled with a liquid, including,for example, an aqueous liquid, a gas, a gaseous precursor, and/or asolid or solute material, including, for example, a bioactive agent, asdesired.

As used herein, the term “vesicular composition” refers to a compositionwhich is formulate from lipids and which comprises vesicles.

As used herein, the term “vesicle formulation” refers to a compositionwhich comprises vesicles and a bioactive agent.

As used herein, the term “lipsomes” refers to a generally sphericalcluster or aggregate of amphipathic compounds, including lipidcompounds, typically in the form of one or more concentric layers, forexample, bilayers. They may also be referred to herein as lipidvesicles.

Angiogenesis is the process of formation of new capillary blood vesselsfrom existing vasculature. It is an important component of a variety ofphysiological processes including ovulation, embryonic development,wound repair, and collateral vascular generation in the myocardium. Itis also central to a number of pathological conditions such as tumorgrowth and metastasis, diabetic retinopathy, and macular degeneration.The process begins with the activation of existing vascular endothelialcells in response to a variety of cytokines and growth factors. Theactivated endothelial cells secrete enzymes that degrade the basementmembrane of the vessels. The endothelial cells then proliferate andmigrate into the extracellular matrix first forming tubules andsubsequently new blood vessels.

Under normal conditions, endothelial cell proliferation is a very slowprocess, but it increases for a short period of time duringembryogenesis, ovulation and wound healing. This temporary increase incell turnover is governed by a combination of a number of growthstimulatory factors and growth suppressing factors. In pathologicalangiogenesis, this normal balance is disrupted resulting in continuedincreased endothelial cell proliferation. Some of the proangiogenicfactors that have been identified include basic fibroblast growth factor(bFGF), angiogenin, TGF-alpha, TGF-beta, and vascular endothelium growthfactor (VEGF), while interferon-alpha, interferon-beta andthrombospondin are examples of angiogenesis suppressors.

Angiogenic factors interact with endothelial cell surface receptors suchas the receptor tyrosine kinases EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1,Tek, Tie, neuropilin-1, endoglin, endosialin, and Axl. The receptorsFlk-1/KDR, neuropilin-1, and Flt-1 recognize VEGF and these interactionsplay key roles in VEGF-induced angiogenesis. The Tie subfamily ofreceptor tyrosine kinases are also expressed prominently during bloodvessel formation.

The proliferation and migration of endothelial cells in theextracellular matrix is mediated by interaction with a variety of celladhesion molecules. Integrins are a diverse family of heterodimeric cellsurface receptors by which endothelial cells attach to the extracellularmatrix, each other and other cells. Angiogenesis induced by bFGF orTNF-alpha depend on the agency of the integrin avb3, while angiogenesisinduced by VEGF depends on the integrin avb5 (Cheresh et. al., Science,1995, 270, 1500–2). Induction of expression of the integrins a1b1 anda2b1 on the endothelial cell surface is another important mechanism bywhich VEGF promotes angiogenesis (Senger, et. al., Proc. Natl. Acad, SciUSA, 1997, 94, 13612–7).

The pharmaceuticals of the present invention are comprised of anon-peptide targeting moiety for the vitronectin receptor that isexpressed or upregulated in angiogenic tumor vasculature.

The ultrasound contrast agents of the present invention comprise aplurality of vitronectin receptor targeting moieties attached to orincorporated into a microbubble of a biocompatible gas, a liquidcarrier, and a surfactant microsphere, further comprising an optionallinking moiety, L_(n), between the targeting moieties and themicrobubble. In this context, the term liquid carrier means aqueoussolution and the term surfactant means any amphiphilic material whichproduces a reduction in interfacial tension in a solution. A list ofsuitable surfactants for forming surfactant microspheres is disclosed inEP0727225A2, herein incorporated by reference. The term surfactantmicrosphere includes nanospheres, liposomes, vesicles and the like. Thebiocompatible gas can be air, or a fluorocarbon, such as a C₃–C₅perfluoroalkane, which provides the difference in echogenicity and thusthe contrast in ultrasound imaging. The gas is encapsulated or containedin the microsphere to which is attached the biodirecting group,optionally via a linking group. The attachment can be covalent, ionic orby van der Waals forces. Specific examples of such contrast agentsinclude lipid encapsulated perfluorocarbons with a plurality of tumorneovasculature receptor binding peptides, polypeptides orpeptidomimetics.

X-ray contrast agents of the present invention are comprised of one ormore vitronectin receptor targeting moieties attached to one or moreX-ray absorbing or “heavy” atoms of atomic number 20 or greater, furthercomprising an optional linking moiety, L_(n), between the targetingmoieties and the X-ray absorbing atoms. The frequently used heavy atomin X-ray contrast agents is iodine. Recently, X-ray contrast agentscomprised of metal chelates (Wallace, R., U.S. Pat. No. 5,417,959) andpolychelates comprised of a plurality of metal ions (Love, D., U.S. Pat.No. 5,679,810) have been disclosed. More recently, multinuclear clustercomplexes have been disclosed as X-ray contrast agents (U.S. Pat. No.5,804,161, PCT WO91/14460, and PCT WO 92/17215).

MRI contrast agents of the present invention are comprised of one ormore vitronectin receptor targeting moieties attached to one or moreparamagnetic metal ions, further comprising an optional linking moiety,L_(n), between the targeting moieties and the paramagnetic metal ions.The paramagnetic metal ions are present in the form of metal complexesor metal oxide particles. U.S. Pat. Nos. 5,412,148, and 5,760,191,describe examples of chelators for paramagnetic metal ions for use inMRI contrast agents. U.S. Pat. Nos. 5,801,228, 5,567,411, and 5,281,704,describe examples of polychelants useful for complexing more than oneparamagnetic metal ion for use in MRI contrast agents. U.S. Pat. No.5,520,904, describes particulate compositions comprised of paramagneticmetal ions for use as MRI contrast agents.

Administration of a compound of the present invention in combinationwith such additional therapeutic agents, may afford an efficacyadvantage over the compounds and agents alone, and may do so whilepermitting the use of lower doses of each. A lower dosage minimizes thepotential of side effects, thereby providing an increased margin ofsafety. The combination of a compound of the present invention with suchadditional therapeutic agents is preferably a synergistic combination.Synergy, as described for example by Chou and Talalay, Adv. EnzymeRegul. 22:27–55 (1984), occurs when the therapeutic effect of thecompound and agent when administered in combination is greater than theadditive effect of the either the compound or agent when administeredalone. In general, a synergistic effect is most clearly demonstrated atlevels that are (therapeutically) sub-optimal for either the compound ofthe present invention, a chemotherapeutic agent or a radiosensitizeragent alone, but which are highly efficacious in combination. Synergycan be in terms of improved tumor response without substantial increasesin toxicity over individual treatments alone, or some other beneficialeffect of the combination compared with the individual components.

The compounds of the present invention, and a chemotherapeutic agent ora radiosensitizer agent, utilized in combination therapy may beadministered simultaneously, in either separate or combinedformulations, or at different times e.g., sequentially, such that acombined effect is achieved. The amounts and regime of administrationwill be adjusted by the practitioner, by preferably initially loweringtheir standard doses and then titrating the results obtained.

The invention also provides kits or single packages combining two ormore active ingredients useful in treating cancer. A kit may provide(alone or in combination with a pharmaceutically acceptable diluent orcarrier), the compound of the present invention and additionally atleast one agent selected from the group consisting of a chemotherapeuticagent and a radiosensitizer agent (alone or in combination with diluentor carrier).

The pharmaceuticals of the present invention have the formulae,(Q)_(d)-L_(n)-(C_(h)—X), (Q)_(d)-L_(n)-(C_(h)—X¹)_(d′),(Q)_(d)-L_(n)-(X²)_(d″), and (Q)_(d)-L_(n)-(X³), wherein Q represents anon-peptide that binds to a receptor expressed in angiogenic tumorvasculature, d is 1–10, L_(n) represents an optional linking group,C_(h) represents a metal chelator or bonding moiety, X represents aradioisotope, X¹ represents paramagnetic metal ion, X² represents aparamagnetic metal ion or heavy atom containing insoluble solidparticle, d″ is 1–100, and X³ represents a surfactant microsphere of anechogenic gas. The interaction of the non-peptide recognition sequencesof the vitronectin receptor binding portion of the pharmaceuticals withthe α_(v)β₃ receptor results in localization of the pharmaceuticals inangiogenic tumor vasculature, which express the α_(v)β₃ receptor.

The pharmaceuticals of the present invention can be synthesized byseveral approaches. One approach involves the synthesis of the targetingnon-peptide moiety, Q, and direct attachment of one or more moieties, Q,to one or more metal chelators or bonding moieties, C_(h), or to aparamagnetic metal ion or heavy atom containing solid particle, or to anechogenic gas microbubble. Another approach involves the attachment ofone or more moieties, Q, to the linking group, L_(n), which is thenattached to one or more metal chelators or bonding moieties, C_(h), orto a paramagnetic metal ion or heavy atom containing solid particle, orto an echogenic gas microbubble. Another approach involves the synthesisof a non-peptide, Q, bearing a fragment of the linking group, L_(n), oneor more of which are then attached to the remainder of the linking groupand then to one or more metal chelators or bonding moieties, C_(h), orto a paramagnetic metal ion or heavy atom containing solid particle, orto an echogenic gas microbubble.

The non-peptide vitronectin binding moieties, Q, optionally bearing alinking group, L_(n), or a fragment of the linking group, can besynthesized using standard synthetic methods known to those skilled inthe art. Preferred methods include but are not limited to those methodsdescribed below.

The attachment of linking groups, L_(n), to the non-peptides, Q;chelators or bonding units, C_(h), to the non-peptides, Q, or to thelinking groups, L_(n); and non-peptides, bearing a fragment of thelinking group to the remainder of the linking group, in combinationforming the moiety, (Q)_(d)-L_(n), and then to the moiety C_(h); can allbe performed by standard techniques. These include, but are not limitedto, amidation, esterification, alkylation, and the formation of ureas orthioureas. Procedures for performing these attachments can be found inBrinkley, M., Bioconjugate Chemistry 1992, 3(1), which is incorporatedherein by reference.

A number of methods can be used to attach the non-peptides, Q, toparamagnetic metal ion or heavy atom containing solid particles, X², byone of skill in the art of the surface modification of solid particles.In general, the targeting moiety Q or the combination (Q)_(d)L_(n) isattached to a coupling group that react with a constituent of thesurface of the solid particle. The coupling groups can be any of anumber of silanes which react with surface hydroxyl groups on the solidparticle surface, as described in co-pending U.S. patent applicationSer. No. 09/356,178, and can also include polyphosphonates,polycarboxylates, polyphosphates or mixtures thereof which couple withthe surface of the solid particles, as described in U.S. Pat. No.5,520,904.

A number of reaction schemes can be used to attach the non-peptides, Q,to the surfactant microsphere, X³. These are illustrated in followingreaction schemes where S_(f) represents a surfactant moiety that formsthe surfactant microsphere.

Acylation Reaction:

Y is a leaving group or active esterDisulfide Coupling:S_(f)—SH+Q-SH→S_(f)—S—S-QSulfonamide Coupling:S_(f)—S(═O)₂—Y+Q-NH₂→S_(f)—S(═O)₂—NH-QReductive Amidation:S_(f)—CHO+Q-NH₂→S_(f)—NH-QIn these reaction schemes, the substituents S_(f) and Q can be reversedas well.

The linking group L_(n) can serve several roles. First it provides aspacing group between the metal chelator or bonding moiety, C_(h), theparamagnetic metal ion or heavy atom containing solid particle, X², andthe surfactant microsphere, X³, and the one or more of the non-peptides,Q, so as to minimize the possibility that the moieties C_(h)—X,C_(h)—X¹, X², and X³, will interfere with the interaction of therecognition sequences of Q with angiogenic tumor vasculature receptors.The necessity of incorporating a linking group in a reagent is dependenton the identity of Q, C_(h)—X, C_(h)—X¹, X², and X³. If C_(h)—X,C_(h)—X¹, X², and X³, cannot be attached to Q without substantiallydiminishing its affinity for the receptors, then a linking group isused. A linking group also provides a means of independently attachingmultiple non-peptides, Q, to one group that is attached to C_(h)—X,C_(h)—X¹, X², or X³.

The linking group also provides a means of incorporating apharmacokinetic modifier into the pharmaceuticals of the presentinvention. The pharmacokinetic modifier serves to direct thebiodistibution of the injected pharmaceutical other than by theinteraction of the targeting moieties, Q, with the vitronectin receptorsexpressed in the tumor neovasculature. A wide variety of functionalgroups can serve as pharmacokinetic modifiers, including, but notlimited to, carbohydrates, polyalkylene glycols, peptides or otherpolyamino acids, and cyclodextrins. The modifiers can be used to enhanceor decrease hydrophilicity and to enhance or decrease the rate of bloodclearance. The modifiers can also be used to direct the route ofelimination of the pharmaceuticals. Preferred pharmacokinetic modifiersare those that result in moderate to fast blood clearance and enhancedrenal excretion.

The metal chelator or bonding moiety, C_(h), is selected to form stablecomplexes with the metal ion chosen for the particular application.Chelators or bonding moieties for diagnostic radiopharmaceuticals areselected to form stable complexes with the radioisotopes that haveimageable gamma ray or positron emissions, such as ^(99m)Tc, ⁹⁵Tc,¹¹¹In, ⁶²Cu, ⁶⁰Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y.

Chelators for technetium, copper and gallium isotopes are selected fromdiaminedithiols, monoamine-monoamidedithiols, triamide-monothiols,monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Thechelators are generally tetradentate with donor atoms selected fromnitrogen, oxygen and sulfur. Preferred reagents are comprised ofchelators having amine nitrogen and thiol sulfur donor atoms andhydrazine bonding units. The thiol sulfur atoms and the hydrazines maybear a protecting group which can be displaced either prior to using thereagent to synthesize a radiopharmaceutical or preferably in situ duringthe synthesis of the radiopharmaceutical.

Exemplary thiol protecting groups include those listed in Greene andWuts, “Protective Groups in Organic Synthesis” John Wiley & Sons, NewYork (1991), the disclosure of which is hereby incorporated byreference. Any thiol protecting group known in the art can be used.Examples of thiol protecting groups include, but are not limited to, thefollowing: acetamidomethyl, benzamidomethyl, 1-ethoxyethyl, benzoyl, andtriphenylmethyl.

Exemplary protecting groups for hydrazine bonding units are hydrazoneswhich can be aldehyde or ketone hydrazones having substituents selectedfrom hydrogen, alkyl, aryl and heterocycle. Particularly preferredhydrazones are described in co-pending U.S. Ser. No. 08/476,296 thedisclosure of which is herein incorporated by reference in its entirety.

The hydrazine bonding unit when bound to a metal radionuclide is termeda hydrazido, or diazenido group and serves as the point of attachment ofthe radionuclide to the remainder of the radiopharmaceutical. Adiazenido group can be either terminal (only one atom of the group isbound to the radionuclide) or chelating. In order to have a chelatingdiazenido group at least one other atom of the group must also be boundto the radionuclide. The atoms bound to the metal are termed donoratoms.

Chelators for ¹¹¹In and ⁸⁶Y are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.Procedures for synthesizing these chelators that are not commerciallyavailable can be found in Brechbiel, M. and Gansow, O., J. Chem. Soc.Perkin Trans. 1992, 1, 1175; Brechbiel, M. and Gansow, O., BioconjugateChem. 1991, 2, 187; Deshpande, S., et. al., J. Nucl. Med. 1990, 31, 473;Kruper, J., U.S. Pat. No. 5,064,956, and Toner, J., U.S. Pat. No.4,859,777, the disclosures of which are hereby incorporated by referencein their entirety.

The coordination sphere of metal ion includes all the ligands or groupsbound to the metal. For a transition metal radionuclide to be stable ittypically has a coordination number (number of donor atoms) comprised ofan integer greater than or equal to 4 and less than or equal to 8; thatis there are 4 to 8 atoms bound to the metal and it is said to have acomplete coordination sphere. The requisite coordination number for astable radionuclide complex is determined by the identity of theradionuclide, its oxidation state, and the type of donor atoms. If thechelator or bonding unit does not provide all of the atoms necessary tostabilize the metal radionuclide by completing its coordination sphere,the coordination sphere is completed by donor atoms from other ligands,termed ancillary or co-ligands, which can also be either terminal orchelating.

A large number of ligands can serve as ancillary or co-ligands, thechoice of which is determined by a variety of considerations such as theease of synthesis of the radiopharmaceutical, the chemical and physicalproperties of the ancillary ligand, the rate of formation, the yield,and the number of isomeric forms of the resulting radiopharmaceuticals,the ability to administer said ancillary or co-ligand to a patientwithout adverse physiological consequences to said patient, and thecompatibility of the ligand in a lyophilized kit formulation. The chargeand lipophilicity of the ancillary ligand will effect the charge andlipophilicity of the radiopharmaceuticals. For example, the use of4,5-dihydroxy-1,3-benzene disulfonate results in radiopharmaceuticalswith an additional two anionic groups because the sulfonate groups willbe anionic under physiological conditions. The use of N-alkylsubstituted 3,4-hydroxypyridinones results in radiopharmaceuticals withvarying degrees of lipophilicity depending on the size of the alkylsubstituents.

Preferred technetium radiopharmaceuticals of the present invention arecomprised of a hydrazido or diazenido bonding unit and an ancillaryligand, A_(L1), or a bonding unit and two types of ancillary A_(L1) andA_(L2), or a tetradentate chelator comprised of two nitrogen and twosulfur atoms. Ancillary ligands A_(L1) are comprised of two or more harddonor atoms such as oxygen and amine nitrogen (sp³ hybridized). Thedonor atoms occupy at least two of the sites in the coordination sphereof the radionuclide metal; the ancillary ligand A_(L1) serves as one ofthe three ligands in the ternary ligand system. Examples of ancillaryligands A_(L1) include but are not limited to dioxygen ligands andfunctionalized aminocarboxylates. A large number of such ligands areavailable from commercial sources.

Ancillary dioxygen ligands include ligands that coordinate to the metalion through at least two oxygen donor atoms. Examples include but arenot limited to: glucoheptonate, gluconate, 2-hydroxyisobutyrate,lactate, tartrate, mannitol, glucarate, maltol, Kojic acid,2,2-bis(hydroxymethyl)propionic acid, 4,5-dihydroxy-1,3-benzenedisulfonate, or substituted or unsubstituted 1,2 or 3,4hydroxypyridinones. (The names for the ligands in these examples referto either the protonated or non-protonated forms of the ligands.)

Functionalized aminocarboxylates include ligands that have a combinationof amine nitrogen and oxygen donor atoms. Examples include but are notlimited to: iminodiacetic acid, 2,3-diaminopropionic acid,nitrilotriacetic acid, N,N′-ethylenediamine diacetic acid,N,N,N′-ethylenediamine triacetic acid, hydroxyethylethylenediaminetriacetic acid, and N,N′-ethylenediamine bis-hydroxyphenylglycine. (Thenames for the ligands in these examples refer to either the protonatedor non-protonated forms of the ligands.)

A series of functionalized aminocarboxylates are disclosed by Bridgeret. al. in U.S. Pat. No. 5,350,837, herein incorporated by reference,that result in improved rates of formation of technetium labeledhydrazino modified proteins. We have determined that certain of theseaminocarboxylates result in improved yields of the radiopharmaceuticalsof the present invention. The preferred ancillary ligands A_(L1)functionalized aminocarboxylates that are derivatives of glycine; themost preferred is tricine (tris(hydroxymethyl)methylglycine).

The most preferred technetium radiopharmaceuticals of the presentinvention are comprised of a hydrazido or diazenido bonding unit and twotypes of ancillary designated A_(L1) and A_(L2), or a diaminedithiolchelator. The second type of ancillary ligands A_(L2) are comprised ofone or more soft donor atoms selected from the group: phosphinephosphorus, arsine arsenic, imine nitrogen (sp² hybridized), sulfur (sp²hybridized) and carbon (sp hybridized); atoms which have p-acidcharacter. Ligands A_(L2) can be monodentate, bidentate or tridentate,the denticity is defined by the number of donor atoms in the ligand. Oneof the two donor atoms in a bidentate ligand and one of the three donoratoms in a tridentate ligand must be a soft donor atom. We havedisclosed in co-pending U.S. Ser. No. 08/415,908, and U.S. Ser. Nos.60/013,360 and 08/646,886, the disclosures of which are hereinincorporated by reference in their entirety, that radiopharmaceuticalscomprised of one or more ancillary or co-ligands A_(L2) are more stablecompared to radiopharmaceuticals that are not comprised of one or moreancillary ligands, A_(L2); that is, they have a minimal number ofisomeric forms, the relative ratios of which do not change significantlywith time, and that remain substantially intact upon dilution.

The ligands A_(L2) that are comprised of phosphine or arsine donor atomsare trisubstituted phosphines, trisubstituted arsines, tetrasubstituteddiphosphines and tetrasubstituted diarsines. The ligands A_(L2) that arecomprised of imine nitrogen are unsaturated or aromaticnitrogen-containing, 5 or 6-membered heterocycles. The ligands that arecomprised of sulfur (sp² hybridized) donor atoms are thiocarbonyls,comprised of the moiety C═S. The ligands comprised of carbon (sphybridized) donor atoms are isonitriles, comprised of the moiety CNR,where R is an organic radical. A large number of such ligands areavailable from commercial sources. Isonitriles can be synthesized asdescribed in European Patent 0107734 and in U.S. Pat. No. 4,988,827,herein incorporated by reference.

Preferred ancillary ligands A_(L2) are trisubstituted phosphines andunsaturated or aromatic 5 or 6 membered heterocycles. The most preferredancillary ligands A_(L2) are trisubstituted phosphines and unsaturated 5membered heterocycles.

The ancillary ligands A_(L2) may be substituted with alkyl, aryl,alkoxy, heterocycle, aralkyl, alkaryl and arylalkaryl groups and may ormay not bear functional groups comprised of heteroatoms such as oxygen,nitrogen, phosphorus or sulfur. Examples of such functional groupsinclude but are not limited to: hydroxyl, carboxyl, carboxamide, nitro,ether, ketone, amino, ammonium, sulfonate, sulfonamide, phosphonate, andphosphonamide. The functional groups may be chosen to alter thelipophilicity and water solubility of the ligands which may affect thebiological properties of the radiopharmaceuticals, such as altering thedistribution into non-target tissues, cells or fluids, and the mechanismand rate of elimination from the body.

Chelators or bonding moieties for therapeutic radiopharmaceuticals areselected to form stable complexes with the radioisotopes that have alphaparticle, beta particle, Auger or Coster-Kronig electron emissions, suchas ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd,¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh,¹¹¹Ag, and ¹⁹²Ir. Chelators for rhenium, copper, palladium, platinum,iridium, rhodium, silver and gold isotopes are selected fromdiaminedithiols, monoamine-monoamidedithiols, triamide-monothiols,monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Chelatorsfor yttrium, bismuth, and the lanthanide isotopes are selected fromcyclic and acyclic polyaminocarboxylates such as DTPA, DOTA, DO3A,2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

Chelators for magnetic resonance imaging contrast agents are selected toform stable complexes with paramagnetic metal ions, such as Gd(III),Dy(III), Fe(III), and Mn(II), are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

The technetium and rhenium radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be easilyprepared by admixing a salt of a radionuclide, a reagent of the presentinvention, an ancillary ligand A_(L1), an ancillary ligand A_(L2), and areducing agent, in an aqueous solution at temperatures from 0 to 100° C.The technetium and rhenium radiopharmaceuticals of the present inventioncomprised of a tetradentate chelator having two nitrogen and two sulfuratoms can be easily prepared by admixing a salt of a radionuclide, areagent of the present invention, and a reducing agent, in an aqueoussolution at temperatures from 0 to 100° C.

When the bonding unit in the reagent of the present invention is presentas a hydrazone group, then it must first be converted to a hydrazine,which may or may not be protonated, prior to complexation with the metalradionuclide. The conversion of the hydrazone group to the hydrazine canoccur either prior to reaction with the radionuclide, in which case theradionuclide and the ancillary or co-ligand or ligands are combined notwith the reagent but with a hydrolyzed form of the reagent bearing thechelator or bonding unit, or in the presence of the radionuclide inwhich case the reagent itself is combined with the radionuclide and theancillary or co-ligand or ligands. In the latter case, the pH of thereaction mixture must be neutral or acidic.

Alternatively, the radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be prepared byfirst admixing a salt of a radionuclide, an ancillary ligand A_(L1), anda reducing agent in an aqueous solution at temperatures from 0 to 100°C. to form an intermediate radionuclide complex with the ancillaryligand A_(L1) then adding a reagent of the present invention and anancillary ligand A_(L2) and reacting further at temperatures from 0 to100° C.

Alternatively, the radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be prepared byfirst admixing a salt of a radionuclide, an ancillary ligand A_(L1), areagent of the present invention, and a reducing agent in an aqueoussolution at temperatures from 0 to 100° C. to form an intermediateradionuclide complex, and then adding an ancillary ligand A_(L2) andreacting further at temperatures from 0 to 100° C.

The technetium and rhenium radionuclides are preferably in the chemicalform of pertechnetate or perrhenate and a pharmaceutically acceptablecation. The pertechnetate salt form is preferably sodium pertechnetatesuch as obtained from commercial Tc-99m generators. The amount ofpertechnetate used to prepare the radiopharmaceuticals of the presentinvention can range from 0.1 mCi to 1 Ci, or more preferably from 1 to200 mCi.

The amount of the reagent of the present invention used to prepare thetechnetium and rhenium radiopharmaceuticals of the present invention canrange from 0.01 μg to 10 mg, or more preferably from 0.5 μg to 200 μg.The amount used will be dictated by the amounts of the other reactantsand the identity of the radiopharmaceuticals of the present invention tobe prepared.

The amounts of the ancillary ligands A_(L1) used can range from 0.1 mgto 1 g, or more, preferably from 1 mg to 100 mg. The exact amount for aparticular radiopharmaceutical is a function of identity of theradiopharmaceuticals of the present invention to be prepared, theprocedure used and the amounts and identities of the other reactants.Too large an amount of A_(L1) will result in the formation ofby-products comprised of technetium labeled A_(L1) without abiologically active molecule or by-products comprised of technetiumlabeled biologically active molecules with the ancillary ligand A_(L1)but without the ancillary ligand A_(L2). Too small an amount of A_(L1)will result in other by-products such as technetium labeled biologicallyactive molecules with the ancillary ligand A_(L2) but without theancillary ligand A_(L1), or reduced hydrolyzed technetium, or technetiumcolloid.

The amounts of the ancillary ligands A_(L2) used can range from 0.001 mgto 1 g, or more preferably from 0.01 mg to 10 mg. The exact amount for aparticular radiopharmaceutical is a function of the identity of theradiopharmaceuticals of the present invention to be prepared, theprocedure used and the amounts and identities of the other reactants.Too large an amount of A_(L2) will result in the formation ofby-products comprised of technetium labeled A_(L2) without abiologically active molecule or by-products comprised of technetiumlabeled biologically active molecules with the ancillary ligand A_(L2)but without the ancillary ligand A_(L1). If the reagent bears one ormore substituents that are comprised of a soft donor atom, as definedabove, at least a ten-fold molar excess of the ancillary ligand A_(L2)to the reagent of formula 2 is required to prevent the substituent frominterfering with the coordination of the ancillary ligand A_(L2) to themetal radionuclide.

Suitable reducing agents for the synthesis of the radiopharmaceuticalsof the present invention include stannous salts, dithionite or bisulfitesalts, borohydride salts, and formamidinesulfinic acid, wherein thesalts are of any pharmaceutically acceptable form. The preferredreducing agent is a stannous salt. The amount of a reducing agent usedcan range from 0.001 mg to 10 mg, or more preferably from 0.005 mg to 1mg.

The specific structure of a radiopharmaceutical of the present inventioncomprised of a hydrazido or diazenido bonding unit will depend on theidentity of the reagent of the present invention used, the identity ofany ancillary ligand A_(L1), the identity of any ancillary ligandA_(L2), and the identity of the radionuclide. Radiopharmaceuticalscomprised of a hydrazido or diazenido bonding unit synthesized usingconcentrations of reagents of <100 μg/mL, will be comprised of onehydrazido or diazenido group. Those synthesized using >1 mg/mLconcentrations will be comprised of two hydrazido or diazenido groupsfrom two reagent molecules. For most applications, only a limited amountof the biologically active molecule can be injected and not result inundesired side-effects, such as chemical toxicity, interference with abiological process or an altered biodistribution of theradiopharmaceutical. Therefore, the radiopharmaceuticals which requirehigher concentrations of the reagents comprised in part of thebiologically active molecule, will have to be diluted or purified aftersynthesis to avoid such side-effects.

The identities and amounts used of the ancillary ligands A_(L1) andA_(L2) will determine the values of the variables y and z. The values ofy and z can independently be an integer from 1 to 2. In combination, thevalues of y and z will result in a technetium coordination sphere thatis made up of at least five and no more than seven donor atoms. Formonodentate ancillary ligands A_(L2), z can be an integer from 1 to 2;for bidentate or tridentate ancillary ligands A_(L2), z is 1. Thepreferred combination for monodentate ligands is y equal to 1 or 2 and zequal to 1. The preferred combination for bidentate or tridentateligands is y equal to 1 and z equal to 1.

The indium, copper, gallium, silver, palladium, rhodium, gold, platinum,bismuth, yttrium and lanthanide radiopharmaceuticals of the presentinvention can be easily prepared by admixing a salt of a radionuclideand a reagent of the present invention, in an aqueous solution attemperatures from 0 to 100° C. These radionuclides are typicallyobtained as a dilute aqueous solution in a mineral acid, such ashydrochloric, nitric or sulfuric acid. The radionuclides are combinedwith from one to about one thousand equivalents of the reagents of thepresent invention dissolved in aqueous solution. A buffer is typicallyused to maintain the pH of the reaction mixture between 3 and 10.

The gadolinium, dysprosium, iron and manganese metallopharmaceuticals ofthe present invention can be easily prepared by admixing a salt of theparamagnetic metal ion and a reagent of the present invention, in anaqueous solution at temperatures from 0 to 100° C. These paramagneticmetal ions are typically obtained as a dilute aqueous solution in amineral acid, such as hydrochloric, nitric or sulfuric acid. Theparamagnetic metal ions are combined with from one to about one thousandequivalents of the reagents of the present invention dissolved inaqueous solution. A buffer is typically used to maintain the pH of thereaction mixture between 3 and 10.

The total time of preparation will vary depending on the identity of themetal ion, the identities and amounts of the reactants and the procedureused for the preparation. The preparations may be complete, resultingin >80% yield of the radiopharmaceutical, in 1 minute or may requiremore time. If higher purity metallopharmaceuticals are needed ordesired, the products can be purified by any of a number of techniqueswell known to those skilled in the art such as liquid chromatography,solid phase extraction, solvent extraction, dialysis or ultrafiltration.

Buffers useful in the preparation of metallopharmaceuticals and indiagnostic kits useful for the preparation of said radiopharmaceuticalsinclude but are not limited to phosphate, citrate, sulfosalicylate, andacetate. A more complete list can be found in the United StatesPharmacopeia.

Lyophilization aids useful in the preparation of diagnostic kits usefulfor the preparation of radiopharmaceuticals include but are not limitedto mannitol, lactose, sorbitol, dextran, Ficoll, andpolyvinylpyrrolidine(PVP).

Stabilization aids useful in the preparation of metallopharmaceuticalsand in diagnostic kits useful for the preparation ofradiopharmaceuticals include but are not limited to ascorbic acid,cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite,gentisic acid, and inositol.

Solubilization aids useful in the preparation of metallopharmaceuticalsand in diagnostic kits useful for the preparation ofradiopharmaceuticals include but are not limited to ethanol, glycerin,polyethylene glycol, propylene glycol, polyoxyethylene sorbitanmonooleate, sorbitan monoloeate, polysorbates,poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers(Pluronics) and lecithin. Preferred solubilizing aids are polyethyleneglycol, and Pluronics.

Bacteriostats useful in the preparation of metallopharmaceuticals and indiagnostic kits useful for the preparation of radiopharmaceuticalsinclude but are not limited to benzyl alcohol, benzalkonium chloride,chlorbutanol, and methyl, propyl or butyl paraben.

A component in a diagnostic kit can also serve more than one function. Areducing agent can also serve as a stabilization aid, a buffer can alsoserve as a transfer ligand, a lyophilization aid can also serve as atransfer, ancillary or co-ligand and so forth.

The diagnostic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of 1 to 100 mCi per 70kg body weight, or preferably at a dose of 5 to 50 mCi. Imaging isperformed using known procedures.

The therapeutic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of 0.1 to 100 mCi per70 kg body weight, or preferably at a dose of 0.5 to 5 mCi per 70 kgbody weight.

The magnetic resonance imaging contrast agents of the present inventionmay be used in a similar manner as other MRI agents as described in U.S.Pat. No. 5,155,215; U.S. Pat. No. 5,087,440; Margerstadt et al., Magn.Reson. Med., 1986, 3, 808; Runge et al., Radiology, 1988, 166, 835; andBousquet et al., Radiology, 1988, 166, 693. Generally, sterile aqueoussolutions of the contrast agents are administered to a patientintravenously in dosages ranging from 0.01 to 1.0 mmoles per kg bodyweight.

For use as X-ray contrast agents, the compositions of the presentinvention should generally have a heavy atom concentration of 1 mM to 5M, preferably 0.1 M to 2 M. Dosages, administered by intravenousinjection, will typically range from 0.5 mmol/kg to 1.5 mmol/kg,preferably 0.8 mmol/kg to 1.2 mmol/kg. Imaging is performed using knowntechniques, preferably X-ray computed tomography.

The ultrasound contrast agents of the present invention are administeredby intravenous injection in an amount of 10 to 30 μL of the echogenicgas per kg body weight or by infusion at a rate of approximately 3μL/kg/min. Imaging is performed using known techniques of sonography.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES

Representative materials and methods that may be used in preparing thecompounds of the (invention are described further below.

Manual solid phase peptide synthesis was performed in 25 mLpolypropylene filtration tubes purchased from BiORad Inc., or in 60 mLhour-glass reaction vessels purchased from Peptides International. Oximeresin (substitution level=0.96 mmol/g) was prepared according topublished procedure (DeGrado and Kaiser, J. Org. Chem. 1980, 45, 1295),or was purchased from Novabiochem (substitution level=0.62 mmol/g). Allchemicals and solvents (reagent grade) were used as supplied from thevendors cited without further purification. t-Butyloxycarbonyl (Boc)amino acids and other starting amino acids may be obtained commerciallyfrom Bachem Inc., Bachem Biosciences Inc. (Philadelphia, Pa.), AdvancedChemTech (Louisville, Ky.), Peninsula Laboratories (Belmont, Calif.), orSigma (St. Louis, Mo.).2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and TBTU were purchased from Advanced ChemTech.N-methylmorpholine (NMM), m-cresol, D-2-aminobutyric acid (Abu),trimethylacetylchloride, diisopropylethylamine (DIEA), 1,2,4-triazole,stannous chloride dihydrate, and tris(3-sulfonatophenyl)phosphinetrisodium salt (TPPTS) were purchased from Aldrich Chemical Company.Bis(3-sulfonatophenyl)phenylphosphine disodium salt (TPPDS) was preparedby the published procedure (Kuntz, E., U.S. Pat. No. 4,248,802).(3-Sulfonatophenyl)diphenylphosphine monosodium salt (TPPMS)waspurchased from TCI America, Inc. Tricine was obtained from ResearchOrganics, Inc. Technetium-99m-pertechnetate (^(99m)TcO₄ ⁻) was obtainedfrom a DuPont Pharma ⁹⁹Mo/^(99m)Tc Technelite® generator.In-111-chloride (Indichlor®) was obtained from Amersham Medi-Physics,Inc. Sm-153-chloride and Lutetium-177-chloride were obtained from theUniversity of Missouri Research Reactor (MURR). Yttrium-90 chloride wasobtained from the Pacific Northwest Research Laboratories.Dimethylformamide (DMF), ethyl acetate, chloroform (CHCl₃), methanol(MeOH), pyridine and hydrochloric acid (HCl) were obtained from Baker.Acetonitrile, dichloromethane (DCM), acetic acid (HOAc), trifluoroaceticacid (TFA), ethyl ether, triethylamine, acetone, and magnesium sulfatewere commercially obtained. Absolute ethanol was obtained from QuantumChemical Corporation.

Synthesis of Boc-Glu-(OTFP)-OTFP

To a solution of Boc-Glu-OH (28.9 g, 117 mmol) in DMF (500 mL) at roomtemperature, and under nitrogen, was added a solution of2,3,5,6-tetrafluorophenol (48.2 g, 290 mmol) in DMF (50 mL). Afterstirring for 10 min. EDC (55.6 g, 290 mmol) was added and the reactionmixture was stirred for about 96 h. The volatiles were removed in vacuoand the residue was triturated in 0.1 N HCl (750 mL). To this mixturewas added ethyl acetate (600 mL), the layers separated. The aqueouslayer was extracted with ethyl acetate (3×˜500 mL), and all the ethylacetate fractions were combined, washed with water (300 mL) and brine(300 mL), dried (MgSO₄), and concentrated to give a tan solid (62 g).The tan solid was washed with acetonitrile to give the title compound(45.5 g, 73%) in purified form.

ESMS: Calculated for C₂₂H₁₇F₈NO₆, 543.09; found, 566.0 [M+Na]⁺¹.

Example 1 Preparation of(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoicacid

Step 1A. Synthesis of tert-butyl3-(((3-((tert-butoxy)carbonylamino)propyl)methylamino)methyl)-4-fluorobenzoate

Crude tert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate (4.6 g., 16mmol), prepared as described in (WO 95/18619, PCT/US95/00248), wasdissolved in 100 mL THF, along with3-tert-butoxycarbonylamino-1-propylamine hydrochloride (2.9 g., 16.6mmol) and diisopropylethylamine added (4.6 g., 36 mmol). The solutionwas stirred overnight, diluted with 1N NaOH, and extracted with threeportions of ether. The combined organics were washed with water and sat.NaCl, dried over MgSO₄, and concentrated under vacuum to 5.7 g. of ayellow oil. This was purified by flash chromatography (CH₂Cl₂/EtOAc) toafford the product as a clear oil (2.04 g., ˜35%). ¹HNMR (600 MHz,DMSO-d6): 7.99 (dd, J=2, 5.1 Hz, 1H), 7.78 (ddd, J=2.3, 2.8, 3.0 Hz,1H), 7.22 (dd, J=8.8, 0.7, 1H), 6.73 (b, 1H), 3.68 (s, 2H), 2.94 (m,2H), 2.15 (b, 1H), 1.51 (s, 9H), 1.49 (m, 2H), 1.33 (s, 9H); MS (ES):765.4 [2M+H]⁺, 383.3 [M+H]⁺.

Step 1B. Synthesis of methyl(S)-3-N-(3-((tert-butoxyl)carbonylamino)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate

The product of Step A (2 g, 5.3 mmol) was dissolved in 20 mL dry DMF,along with N-Cbz-L-aspartic acid β-methyl ester (1.65 g, 5.9 mmol), and1-hydroxybenzotriazole hydrate (800 mg, 5.9 mmol) under a nitrogenatmosphere. Dicyclohexylcarbodiimide (1M in CH₂Cl₂, 5.9 mL, 5.9 mmol)was added via syringe, and the solution stirred 18 hr. Ether (25 mL) wasadded and the solids were filtered and rinsed with ether. The filtratewas concentrated, redissolved in ether, filtered, and the filtratewashed with sat. bicarbonate, water, and sat. NaCl. It was dried(Na2SO4), filtered and concentrated to a yellow oil which was purifiedby flash chromatography (4:1 CH2Cl2/EtOAc) to afford the product (3.0 g,87%) as a clear oil. ¹HNMR (600 MHz, DMSO-d6): mixture of amiderotamers: 7.82 (m, 2H), 7.71 (m, 1H), 7.3 (m, 6H), 6.72 (bd, 1H), 5:02(dd, J=12.5, 25.7 Hz, 1H), 4.44–4.88 (m, 4H), 3.52 (d, 2H), 3.27 (d,3H), 3.10–3.45 (m, 4H) 2.45–2.90 (m, 4H), 1.55 (m, 2H), 1.49 (s, 9H),1.31 (s, 9H); MS-ES: 590.3 [(M-tBu)+H]⁺, 646.4 [M+H]⁺, 668.4 [M+Na]⁺.

Step 1C: Synthesis of methyl(S)-3-amino-3-(N-(3-((tert-butoxy)carbonylamino)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)propanoate

The product of step B (2.8 g, 4.4 mmol) was dissolved in MeOH (50 mL)with 10% Pd/C (530 mg) and shaken under a hydrogen atmosphere (50 psi)in a Parr shaker for 2 hr. The reaction mixture was filtered throughCelite® and concentrated to a clear oil (2.14 g, 94%) under vacuum,which was not further purified. MS-ES: 512.4 [M+H]⁺, 1023.5 [2M+H]⁺;

Step 1D: Synthesis of methyl(S)-2-(2,5-diaza-9-((tert-butyl)oxycarbonyl)-5-(3-((tert-butoxy)carbonylamino)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The crude oil from C (2.14 g, 4.0 mmol) was dissolved in dryN-methylpyrollidinone (50 mL) along with 2,6-di-tert-butylpyridine (2.1mL, 9.2 mmol) under nitrogen. The solution was heated at 125° C. in anoil bath for 43 hours. The solution was cooled, poured into 100 mLwater, and extracted with ethyl acetate. The organics were concentratedto an oil and purified by flash chromatography (CH₂Cl₂/EtOAc) to afford1.0 g (46%) of the product. MS-ES: 392.3 [(M-tBoc)+H]⁺ 436.3[(M-tBu)+H]⁺ 492.4 [M+H]⁺, 983.6 [2M+H]⁺;

Step 1E: Synthesis of(S)-2,5-diaza-5-(3-((tert-butoxy)carbonylamino)propyl)-3-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-9-carboxylicacid

The ester from D (880 mg, 1.8 mmol) was dissolved in dichloromethane (12mL) and trifluoroacetic acid (6 mL) added with stirring under nitrogen.The reaction was stirred 2 hours, concentrated under vacuum, andredissolved in 7 mL dichloromethane. Acetonitrile (7 mL) was added,followed by di-tert-butyldicarbonate (590 mg, 2.7 mmol) anddiisopropylethylamine (1.4 mL, 7.6 mmol). The reaction was stirredovernight under nitrogen. EtOAc (15 mL) was added and the entiresolution was washed with 5% citric acid and brine, dried (MgSO4), andconcentrated to 1.12 g of oil. This was purified by flash chromatography(CH2Cl2/EtOAc/MeOH) and the residue dissolved in 0.1% TFA/acetonitrile(50 mL) and lyophilized to afford the product (680 mg, 69%) as a whitepowder. ¹HNMR (600 MHz, DMSO-d6): 12.14 (b, 1H), 7.62 (d, J=1.8 Hz, 1H),7.53 (dd, J=1.9 Hz, 8.5 Hz, 1H), 6.66 (bt, J=5.4 Hz, 1H), 6.56 (d, J=8.5Hz, 1H), 6.55 (m, 1H) 5.41 (d, J=16.6 Hz) 1H), 5.15 (dd, J=5 Hz, 8.8 Hz,1H), 4.02 (d, 16.7 Hz, 1H), 3.60 (s, 3H), 3.38 (m, 2H), 2.84 (m, 2H),2.82 (dd, J=8.8 Hz, 16.6 Hz, 1H), 2.67 (dd, J=5.3 Hz, 16.6 Hz, 1H), 1.50(m, 2H), 1.36 (s, 9H); LRMS(ES): 380.3 [(M-tBu)+H]⁺, 436.3 [M+H]⁺.

Step 1F. Synthesis of methyl(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(3-((tert-butoxy)carbonylamino)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of step 1E (476 mg, 1.09 mmol) was dissolved in drydimethylformamide along with 2-(methylaminomethyl)benzimidazoledihydrochloride (290 mg, 1.25 mmol, prepared according to F. Ali et.al., WO 96/00730), hydroxybenzotriazole hydrate (HOBT) (154 mg, 1.14mmol), ethyl dimethylaminopropylcarbodiimide hydrochloride (261 mg, 1.36mmol), and diisopropylethylamine (1.1 mL, 6 mmol). The solution wasstirred for 23 hr under nitrogen and then concentrated. The residue waspartitioned with ethyl acetate/water, and the aqueous layer extractedwith 2 portions of ethyl acetate. The combined organic layers werewashed with water and brine and concentrated. The residue was purifiedby flash chromatography on silica (95:5 ethyl acetate/methanol) and theproduct fractions concentrated to afford the product (435 mg, 69%) as acrunchy foam after drying under vacuum. LRMS(ES): 579.4 [(M+H]⁺. ¹HNMR(600.1300 MHz, DMSO-d6): 12.34 (b, 1H), 7.58 (d, J=1.8 Hz, 1H), 7.48(dd, J=1.9 Hz, 8.5 Hz, 1H), 7.24 (s, 1H), 7.17 (m, 3H), 6.64 (t, 1H),6.56 (d, 1H), 6.55 (m, 1H) 6.21 (s, 1H), 5.41 (d, J=16.6 Hz, 1H), 5.10(dd, J=5 Hz, 8.8 Hz, 1H), 4.76 (q, 2H), 3.89 (d, 16.6 Hz, 1H), 3.60 (s,3H), 3.37 (m, 2H), 3.04 (s, 3H), 2.82 (m, 3H), 2.64 (dd, J=5.3 Hz, 16.6Hz, 1H), 1.48 (m, 2H), 1.34 (s, 9H).

Step 1G: Synthesis of(S,S)-7-((tert-butyl)oxycarbonyl)-2-(2-((tert-butyl)oxycarbonyl)ethyl)-3-oxo-5-((phenylmethoxy)carbonylamino)carbonyl)heptanoicacid

Gamma-tert-butoxy-z-glutamic acid succinimide ester (2.0 g, 4.75 mmol)was dissolved in dimethylformamide, and gamma-tert-butoxyglutamic acid(0.98 g, 4.8 mmol) added, followed by diisopropylethylamine (1.75 mL,10.1 mmol). The solution was stirred 18 hr, concentrated, and theresidue partitioned into ethyl acetate/10% citric acid. The aqueousfraction was extracted with ethyl acetate and the combined organics werewashed with water, 10% potassium hydrogen sulfate, and brine, and thenconcentrated. The residual oil was purified by flash chromatography onsilica (CH₂Cl₂/EtOAc/EtOH, 1:1:0.5%) and the product fractions combinedand evaporated to yield the product (1.3 g, 53%) as a gummy solid. LRMS(ES): 523.4 [M+H]⁺, 467.4; ¹HNMR (600.1330 MHz, CDCl₃) 7.30 (m, 6H),5.80 (d, 1H), 5.09 (m, 2H), 4.53 (m, 1H), 4.29 (m, 1H), 2.36 (m, 4H),1.88–2.16 (m, 4H), 1.42 (s, 9H), 1.41 (s, 9H).

Step 1H: Synthesis of tert-butyl(S,S,S)-4-(N-(3-(3,6-diaza-5-((methoxycarbonyl)methyl)-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxycarbonylamino)butanoylamino)butanoate

The product of 1F (40 mg, 70 μmol) was dissolved in dichloromethane (1mL) under nitrogen. To this was added triethylsilane (110 uL, 0.7 mmol)and trifluoroacetic acid (1 mL). The reaction was stirred 60 min,concentrated, and reconcentrated with 5 mL toluene. The residue wasdissolved in dry dimethylformamide (1 mL) and the product of step 1G (40mg, 77 μmol) added, along with HBTU (33.2 mg, 87 μmol) anddiisopropylethylamine (100 μL, 560 μmol). This was stirred for 18 hr.The reaction was concentrated, and the residue dissolved in ethylacetate. The organics were washed with water, 10% potassium hydrogensulfate, water, and brine, and then concentrated. The residual oil waspurified by flash chromatography on silica (EtOAc/2-PrOH, 1%→10%) andthe product fractions combined and evaporated to yield the product (36mg, 53%) as a white solid. LRMS (ES): 983.6 [M+H]⁺, 492.5 [M+2H]⁺²; HRMS(ESI): Calculated for C₅₁H₆₇N₈O₁₂—983.4878, found—983.4860; ¹HNMR(600.1300 MHz, CDCl₃) 7.63 (b, 2H), 7.45 (b, 1H) 7.22–7.41 (m, 11H),6.90 (b, 1H), 6.54 (d, 1H), 5.99 (b, 1H) 5.39 (d, J=16.6 Hz, 1H), 5.12(m, 3H), 4.78–4.98 (m, 2H), 4.51 (b, 1H), 4.40 (b, 1H), 4.25 (b, 1H),3.87 (d, J=16.6 Hz 1H), 3.76 (s, 3H), 3.66 (b, 1H), 3.45 (b, 1H), 3.19(s, 3H), 3.17 (m, 1H), 3.03 (m, 2H), 2.69 (dd, 1H), 2.25–2.45 (m, 4H)2.05–2.16 (m, 2H), 1.96 (m, 2H), 1.71 (m, 2H), 1.46 (s, 9H), 1.44 (s,9H).

Step 1I: Synthesis of tert-butyl(S,S,S)-4-amino-4-(N-(3-(3,6-diaza-5-((methoxycarbonyl)methyl)-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoateacetate salt

The product of Step 1H (33 mg, 33 μmol) was hydrogenated with 10%palladium on carbon (15 mg) in methanol (6 mL) with acetic acid (0.1 mL)on a Parr shaker at 40 psi for 1.5 hr. The solution was filtered onCelite, rinsed with methanol and concentrated. The residue was dissolvedin 20 mL 1:1 acetonitrile/water, frozen, and lyophilized to afford theproduct as a white powder (21 mg, 75%). LRMS (ES): 849.5 [M+H]⁺, 425.5[M+2H]⁺²;

Step J: Synthesis of tert-butyl(S,S,S)-4-(N-(1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,5,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoatetrifluoroacetate

The product of step 1I (20 mg, 16.8 μmol) was dissolved in DMF (1 mL)along with DOTA(OtBu)3-OH (26 mg, 25 μmol), HBTU (20 mg, 53 μmol),diisopropylethylamine (29.1 mg, 225 μmol) and HOBT hydrate (2.5 mg, 18μmol). This was stirred for 18 hr under nitrogen, concentrated undervacuum, and purified by preparative HPLC (Vydac C-18, 2.5 cm×15 cm, 0.1%TFA/acetonitrile gradient). The product fractions were pooled andlyophilized to afford 17.5 mg of product as a white powder. LRMS (ES)589.5, 617.8, 646.1, 674.5 [(M-ntBu) +2H]+2, 702.8 [M+2H]+2, 1403.9[M+H]⁺

Step 1K: Synthesis of(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoicacid

The product of I (16 mg, 7.67 μmol (as 6TFA salt)) was dissolved inTHF/MeOH (1:1, 1 mL) and lithium hydroxide added (26 μL of a 3M solutionin water). The reaction was stirred for 2 hr, concentrated, and treatedwith trifluoroacetic acid (0.8 mL) and triethylsilane (0.2 mL) undernitrogen. The solution was stirred for 21 hr, concentrated under vacuum,and purified by preparative HPLC (Vydac C-18, 21.5 mm×15 cm, 0.1%TFA/acetonitrile gradient). The product fractions were pooled andlyophilized to afford the product (6.5 mg, 55%) as a white powder. LRMS(ES): 370.9 [M+3H]+3, 555.6 [M+2H]+2, 1109.5 [M+H]+; HRMS: Calculatedfor C₅₀H₆₉O₁₇N₁₂: 1109.4904, found: 1109.4890.

Example 2 Preparation of(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid trifluoroacetate salt

Step 2A: Synthesis of tert-butyl3-(((6-((tert-butoxy)carbonylamino)hexyl)amino)methyl)-4-fluorobenzoate

This was prepared in the same fashion as Example 1A fromtert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate (5.4 g., 18 mmol) and6-tert-butoxycarbonylamino-1-hexylamine hydrochloride (5.0 g., 19.8mmol), affording 3.1 g (41%) of product as a yellow oil. LRMS: 425.2[M+H]⁺; ¹HNMR (270 MHz, DMSO-d6): 7.95 (dd, 1H), 7.87 (dd, 1H), 7.04 (t,1H), 4.50 (bs, 1H), 3.83 (s, 2H), 3.07 (q, 2H), 2.59 (t, 2H), 1.57 (s,9H), 1.42 (s, 9H), 1.60–1.20 (m, 8H);

Step 2B: Synthesis of methyl(S)-3-N-(6-((tert-butoxyl)carbonylamino)hexyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate

This was prepared as in Example 1B, starting with 3.06 g of amine,affording 4.4 g (88%) of the product as a viscous oil. LRMS: 688.4[M+H]⁺; ¹HNMR (270 MHz, DMSO-d6): Mixture of amide rotamers, 7.85 (m,2H), 7.80 (d, 1H), 7.4–7.2 (m, 6H), 6.73 (br t, 1H), 5.10–4.40 (m, 4H),3.56, 3.53 (2s, 3H), 3.35 (m, 2H), 3.00–2.55 (m, 4H), 1.51 (s, 9H), 1.35(s, 9H), 1.70–1.10 (m, 8H);

Step 2C: Synthesis of methyl(S)-3-amino-3-(N-(6-((tert-butoxy)carbonylamino)hexyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)propanoate

This step was done in the same fashion as Example 1C, starting with 2.3g of CbZ protected compound, affording 1.71 g (92%) of the amine as apale yellow oil. LRMS: 554.3 [M+H]⁺; ¹HNMR (270 MHz, DMSO-d6) mixture ofamide rotamers: 7.90–7.70 (m, 2H), 7.29 (m, 1H), 6.75 (br, 1H), 4.80 (q,1H), 4.54 (s, 2H), 4.10 (q, 1H), 3.89 (2t, 1H), 3.53 (2s, 3H) 2.87 (m,2H), 2.55 (m, 2H), 1.90 (bs, 1H), 1.52 (s, 9H), 1.35 (s, 9H), 1.70–1.10(m, 8H);

Step 2D: Synthesis of methyl(S)-2-(2,5-diaza-9-((tert-butyl)oxycarbonyl)-5-(6-((tert-butoxy)carbonylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

This step was done in the same fashion as Example 1D, starting with 1.66g of amine, affording 706 mg (44%) of the benzodiazepine as a paleyellow foam. LRMS: 534.3 [M+H]⁺; ¹HNMR (270 MHz, DMSO-d6) mixture ofamide rotamers: 7.55 (d, 1H), 7.50 (dd, 1H), 6.70 (br t, 1H), 6.55 (br,1H), 6.54 (d, 1H), 5.40 (d, 1H), 5.14 (m, 1H), 3.99 (d, 1H), 3.59 (s,3H) 2.78 (m, 2H), 2.65 (q, 2H), 1.49 (s, 9H), 1.35 (s, 9H), 1.30–1.00(m, 8H);

Step 2E: Synthesis of(S)-2,5-diaza-5-(6-((tert-butoxy)carbonylamino)hexyl)-3-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-9-carboxylicacid

This step was done in the same fashion as Example 1E, starting with 301mg of ester, affording the crude product (394 mg) as a yellow foam,which was used directly in the next step without purification. LRMS:478.2 [M+H]⁺.

Step 2F: Synthesis of methyl(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-((tert-butoxy)carbonylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The reaction was carried out as in Example 1F, obtaining 306 mg of crudesolid, which was further purified by flash chromatography to afford thedesired product (164 mg, 47% from Step D) as a pale yellow solid. LRMS:621.3 [M+H]⁺; ¹HNMR (270 MHz, DMSO-d6): 12.40 (br, 1H), 7.53 (bs, 2H),7.20 (m, 4H), 6.71 (br, 1H), 6.52 (d, 1H), 6.23 (bd, 1H), 5.40 (d, 1H),5.10, (m, 1H), 4.76 (s, 2H), 3.85 (bd, 1H), 3.59 (s, 3H), 3.04 (s, 3H),2.90–2.55 (m, 2H), 1.35 (s, 9H), 1.40–1.20 (m, 8H).

Step 2G: Synthesis of(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-((tert-butoxy)carbonylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

The product of step F (152 mg, 245 μmol) was stirred with lithiumhydroxide (21 mg, 500 μmol) in THF/H2O (3 mL/2 mL) for 22 hr. THF wasremoved under vacuum, the residue diluted with water and acidified withsolid citric acid. The precipitated solid and solution was extractedwith dichloromethane, washed with brine, dried (Na₂SO₄), andconcentrated to afford the acid product (120 mg, 81%) as a pale yellowpowder, which was not purified further. LRMS: 607.2 [M+H]⁺.

Step 2H: Synthesis of(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid trifluoroacetate

The product of step G (87 mg, 143 μmol) was dissolved in CH₂Cl₂ (4 mL)and trifluoroacetic acid (2 mL) added with stirring under nitrogen. Thesolution was stirred for one hour, concentrated under vacuum, and theresidue redissolved in dry DMF (2.5 mL). To this was added sodium2-[[[5-[[(2,5-dioxo-1-pyrollidinyl)oxy]carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonate(75 mg, 170 μmol) and diisopropylethylamine (500 μL, 2.87 mmol)withstirring under nitrogen. The reaction was stirred overnight,concentrated, and the residue purified by preparative HPLC (Vydac C-18,2.5 cm×15 cm, 0.1% TFA/acetonitrile gradient). The product fractionswere combined and lyophilized to afford the product as a pale yellowpowder (47.3 mg, 35%). LRMS (ES): 810.3 [M+H]⁺. ¹HNMR (600.1300 MHz,DMSO-d6): 12.40 (b, 2H), 9.24 (bs, 1H), 8.59 (bs, 1H), 8.50 (s, 1H),8.24 (bs, 1H), 8.20 (bs, 1H), 7.80 (d, 3H), 7.53 (m, 2H), 7.41 (m, 2H),7.20 (m, 3H), 6.57 (d, 1H), 6.32 (bs, 1H), 5.40 (d, 1H, J=16.4 Hz), 5.10(m, 1H), 4.76 (s, 2H), 3.85 (d, 1H, J=16.4 Hz), 3.55 (m, 2H), 3.21 (m,2H), 3.04 (s, 3H), 2.79 (dd, 1H, J=16.5 Hz, 9 Hz), 2.55 (dd, 1H, J=16.5Hz, 5 Hz), 1.60 (m, 2H), 1.51 (m, 2H), 1.26 (m, 2H), 1.19 (m, 2H).

Example 3 Synthesis of(S)-2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid trifluoroacetate

Step 3A: Synthesis ofN-(6-((benzimidazol-2-ylmethyl)amino)hexyl)(phenylmethoxy)formamidedihydrochloride

Both α-bromomethyl-(N-tert-butoxycarbonyl)benzimidazole (3.42 g, 11mmol, prepared according to WO96/00730) andN-(mono-benzyloxycarbonyl)-hexanediamine (4.58 g, 16 mmol, preparedaccording to Bioconj. Chem., 1997, 8, 611) were dissolved in THF (100mL), along with diisopropylethylamine (8 mL, 45.9 mmol) and water (3mL). The mixture was stirred for 20 hr, concentrated, and the residuepartitioned between 1N NaOH and dichloromethane. The aqueous wasreextracted and concentrated to afford a yellow semi-solid product whichwas dissolved in ether/dichloromethane (2:1, 300 mL) and treated with 4NHCl in dioxane (40 mL, 160 mmol) with stirring at room temperature for18 hr. The resulting solids were filtered, dissolved in a minimum amountof 10% sodium carbonate, extracted into dichloromethane and concentratedto an oil. This was purified by flash chromatography on silica (9:1EtOAc/EtOH, 0.1% NH₄OH) and the product fractions concentrated,dissolved in ether, and treated with 4N HCl/dioxane. The resultingsolids were filtered and washed with ether to afford 745 mg of a whitepowder. LRMS: 381.3 [M+H]⁺; ¹HNMR (270 MHz, DMSO-d6): 10.04 (b, 2H),7.78 (m, 2H), 7.44 (m, 2H), 7.34 (m, 6H) 6.76 (b, 2H), 4.99 (s, 2H),4.60 (s, 2H), 3.10 (m, 2H), 2.99 (m, 2H), 1.67 (m, 2H), 1.41 (m, 2H),1.29 (m, 4H)

Step 3B: Synthesis of methyl(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-(6-((phenylmethoxy)carbonylamino)hexyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of Step 3A (300 mg, 0.66 mmol), methyl(S)-(−)-7-carboxy-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetate(172 mg, 0.55 mmol, prepared according to PCT/US95/00248, WO 95/18619),HOBT (89 mg, 0.66 mmol), and diisopropylethylamine (380 μL, 2.18 mmol)were dissolved in dry DMF (5 mL) in dry glassware under nitrogen. EDC(89 mg, 0.66 mmol) was added in one portion and the reaction stirred 20hr. The solution was concentrated, partitioned between water and ethylacetate, and the aqueous layer extracted with two additional portions ofethyl acetate. The combined organics were washed with water and brine,and concentrated. The crude oil was purified by flash chromatography onsilica gel (EtOAc, 0.5% EtOH). The product fractions were combined andconcentrated to yield 145 mg (40%) of product as a light brown solid.LRMS (ES): 655.3 [M+H]⁺; ¹HNMR (600.1343 MHz, DMSO-d6): 12.38 (b, 1H),7.51 (m, 2H), 7.30 (m, 6H), 7.14 (m, 4H), 6.51 (d, 1H), 6.16 (d, 1H),5.42 (d, 1H, J=16 Hz), 5.08 (m, 1H), 4.96 (s, 2H), 4.73 (s, 2H), 3.88(d, 1H, J=16 Hz), 3.57 (s, 3H), 3.33 (m, 2H), 2.89 (m, 2H), 2.85 (s,3H), 2.78 (dd, 1H, J=16.5 Hz, 9 Hz), 2.61 (dd, J=16.5 Hz, 5 Hz), 1.52(m, 2H), 1.30 (m, 2H), 1.15 (m, 4H); ¹³C NMR (600.1343 MHz, DMSO-d6):170.9, 169.1, 165.6, 156.0, 151.3, 147.4, 137.3, 129.3, 128.3, 127.8,127.7, 127.3, 123.0, 118.1, 114.9, 65.0, 59.7, 51.6, 51.3, 50.1, 50.0,37.4, 35.0, 29.5, 29.2, 26.6, 20.7, 14.1

Step 3C: Synthesis of methyl(S)-2-(9-(N-(6-aminohexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-2,5-diaza-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of 3B (140 mg, 214 μmol) was dissolved in methanol (6 mL)with 10% palladium on carbon (30 mg). The slurry was hydrogenated at oneatmosphere pressure for 5.5 hr, filtered through Celite® andconcentrated to yield the product (100 mg, 90%) as a clear oil which wasnot further purified, but taken directly into the next step. LRMS (ES)521.4 [M+H]⁺, 275.3, 261.3, 245.2, 231.3.

Step 3D: Synthesis of(S)-2-(9-(N-(6-aminohexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-2,5-diaza-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

The product of Step 3C (100 mg, 192 μmol) was dissolved inmethanol/tetrahydrofuran (2:1, 1 mL) and lithium hydroxide hydrate (23mg, 550 μmol) dissolved in 0.5 mL water was added. The reaction wasstirred for 4 hr, neutralized with 10% potassium hydrogen sulfatesolution, and concentrated. The solids were dissolved in methanol,filtered, and the filtrate concentrated to an oil, which was dissolvedin water/acetonitrile and lyophilized to afford 93 mg (96%) of theproduct as a white solid. LRMS (ES): 507.3 [M+H]⁺, 459.4, 254.4[M+2H]⁺²; ¹HNMR (600.1300 MHz, DMSO-d6): 12.35 (b, 1H), 10.49 (b, 3H),7.59 (m, 2H), 7.53 (m, 2H), 7.16 (bs, 4H), 6.53 (d, 1H, J=7.4 Hz), 6.18(s, 1H), 5.44 (d, 1H, J=16.4 Hz), 5.08 (m, 1H), 4.76 (s, 2H), 3.80 (bd,1H, J=12 Hz), 3.38 (m, 2H), 2.88 (s, 3H), 2.78 (dd, 1H, J=16.7 Hz, 9Hz), 2.71 (m, 2H), 2.61 (dd, 1H, J=16.7 Hz, 5 Hz), 1.55 (m, 2H), 1.47(m, 2H), 1.18 (m, 2H), 1.03 (m, 2H)

Step 3E: Synthesis of2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid trifluoroacetate

The product of Step D (80 mg, 160 μmol) was dissolved in drydimethylformamide, along with sodium2-[[[5-[[(2,5-dioxo-1-pyrolidinyl)oxy]carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonate(88 mg, 250 μmol) and diisopropylethylamine (280 μL, 1.6 mmol) withstirring under nitrogen. The reaction was stirred overnight,concentrated, and the residue purified by preparative HPLC (Vydac C-18,21.5 mm×25 cm, 0.1% TFA/acetonitrile gradient). The product fractionswere combined and lyophilized to afford the product as a white solid (24mg, 18%). LRMS (ES): 810.3 [M+H]⁺, 4764.3, 39.9.3; HRMS (ESI):Calculated for C₄₀H₄₄N₉O₈S (M+H)—810.3033, found—810.3052. ¹HNMR(600.1300 MHz, DMSO-d6): 12.40 (b, 2H), 9.24 (bs, 1H), 8.59 (bs, 1H),8.50 (s, 1H), 8.24 (bs, 1H), 8.20 (bs, 1H), 7.80 (d, 3H), 7.53 (m, 2H),7.41 (m, 2H), 7.20 (m, 3H), 6.57 (d, 1H), 6.32 (bs, 1H), 5.47 (d, 1H,J=16.4 Hz), 5.08 (m, 1H), 4.98 (s, 2H), 3.83 (d, 1H, J=16.4 Hz), 3.50(m, 2H), 3.21 (m, 2H), 2.89 (s, 3H), 2.75 (dd, 1H, J=16.7 Hz, 9 Hz),2.53 (dd, 1H, J=16.7 Hz, 5 Hz), 1.65 (m, 2H), 1.48 (m, 2H), 1.26 (m,2H), 1.19 (m, 2H)

Example 4 Preparation of(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonicacid

Step 4A. Synthesis of(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

The product of Step 2E (350 mg, 564 μmol) was dissolved inmethanol/tetrahydrofuran (2:1, 8 mL) with stirring. Lithium hydroxidehydrate (95 mg, 2.25 mmol) was dissolved in water (5 mL) and added tothis solution. It was stirred for two hours, neutralized with 10%potassium hydrogen sulfate and concentrated to a gummy solid. This wasadded to a solution of trifluoroacetic acid in dichloromethane (4 mL/6mL) and stirred for two hours. The solids were filtered off, and thefiltrate concentrated to afford an oil, which was redissolved inwater/acetonitrile and lyophilized to a white powder which was notfurther purified. LRMS (ES): 507.4 [M+H]⁺, 254.4 [M+2H]⁺².

Step 4B. Synthesis of(S,S)-2-(2,5-diaza-(9-(N-benzimidazol-2-ylmethyl))-5-(6-(4-(N-(6-(3,6-diaza-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-2-((tert-butoxy)carbonylamino)butanoylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(11),7(8),9-trien-3-yl)aceticacid

The product of 4A (31 mg, 36.5 μmol) was dissolved in drydimethylformamide (1.5 mL), along with diisopropylethylamine (51 μL, 300μmol). To this was addedbis-(N-hydroxysuccinimide)-N-(tert-butoxycarbonyl)-glutamate (7.7 mg,17.5 μmol) with stirring. The solution was allowed to stir for threehours, when it was concentrated and purified by preparative HPLC (vydacC-18, 21.5 mm×25 cm, 0.1% TFA/acetonitrile gradient). The productfractions were combined and lyophilized to afford the product as a whitesolid (12 mg, 33%). LRMS (ES): 1224.7 [M+H]⁺, 613.1 [M+2H]⁺², 409.3[M+3H]⁺³. HRMS (ESI): Calculated for C₆₄H₈₂N₁₃O₁₂—1224.6206,found—1224.619.

Step 4C. Synthesis of(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl)benzenesulfonicacid

The product of 4B (10 mg, 5.5 μmol of 4TFA salt) was dissolved indichloromethane:triflouroacetic acid (1.5 mL/0.5 mL) under nitrogen. Itwas stirred 20 minutes and concentrated to an oil, which was resuspendedin toluene and reconcentrated to remove residual TFA. The residue wastreated as in step 3E to afford 2.5 mg (31%) of the product as a whitelyophilized solid. LRMS (ES): 1428.2 [M+H]⁺, 714.5 [M+2H]⁺², 477.3[M+3H]⁺³. HRMS (ESI): Calculated for C₇₂H₈₃N₁₆O₁₄S—1427.5995,found—1427.601.

Example 5 Preparation of(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)butanoicacid

Step 5A. Synthesis ofbenzyl((1-(triphenylmethyl)imidazol-2-yl)methyl)amine

N-tritylimidazole-2-carboxaldehyde (338 mg, 1 mmol, prepared accordingto K. L. Kirk; J. Org. Chem., 1978, 43, 4381) was dissolved in drytoluene (7 mL) and anhydrous magnesium sulfate (602 mg, 5 mmol) addedwith stirring under nitrogen. Benzylamine (131 μL, 1.2 mmol) was addedand the solution stirred for 3.5 hr. The solids were filtered undernitrogen and the reaction concentrated. The residue is redissolved in1,2-dichloroethane (25 mL) and cooled to 0° C. Sodiumtriacetoxyborohydride (1.06 g, 5 mmol) was added slowly. The solutionwas allowed to warm to room temperature over 2.5 hours. The reactionmixture was added to water/ethyl acetate and the layers separated. Theaqueous layer was extracted with two portions of ethyl acetate and thecombined organic layers washed with sat. bicarbonate, water, and brine.The solution was concentrated to an oil and purified by flashchromatography on silica gel (99:1 EtOAc/EtOH with 0.1% triethylamine)to afford 330 mg (77%) of product as an oil which solidified onstanding. LRMS (ES): 430.4 [M+H]⁺, 243.2; ¹HNMR (600.1328 MHz, DMSO-d6):7.37 (m, 11H), 7.04 (m, 9H), 6.92 (d, 1H), 6.64 (d, 1H), 3.34 (s, 2H),2.77 (2H).

Step 5B. Synthesis of methyl(S)-2-(2,5-diaza-5-(3-((tert-butoxy)carbonylamino)propyl)-4-oxo-9-(N-benzyl-N-((2-(triphenylmethyl)imidazol-2-yl)methyl)carbamoyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of step 5E (150 mg, 0.345 mmol) was treated in the samemanner as step 1F, affording the product (250 mg, 85%) as a thick oil.LRMS (ES): 847.5 [M+H]⁺, 430.5, 243.2; ¹HNMR (600.1330 MHz, CDCl₃) Thissample gave broad peaks with little fine splitting, even whenrefiltered, and was qualitatively similar to 1E for the benzodiazepinenucleus.

Step 5C. Synthesis of methyl(S)-2-(5-(3-aminopropyl)-2,5-diaza-9-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of step 5B (220 mg, 0.26 mmol) was added to neattrifluoroacetic acid (4 mL) containing triethylsilane (1 mL) undernitrogen and stirred for 1.5 hr. The solution was concentrated andresidual acid removed by reconcentration with toluene. This product wasnot purified, but was used directly in the following step. LRMS (ES):505.4 [M+H]⁺, 253.4.

Step 5D. Synthesis of tert-butyl(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

A portion of the product of step 5C (65 mg, 130 μmol) was reacted withstep 1G as in Step 1H to afford the product (64 mg, 49% from 5B) as anoil. LRMS (ES): 1009.7 [M+H]⁺, 505.6 [M+2H]⁺²,; HRMS (ESI): Calculatedfor C₅₃H₆₉N₈O₁₂—1009.5035, found—1009.502; ¹HNMR (600.1330 MHz, CDCl₃)7.47 (b, 1H), 7.22–7.41 (m, 14H), 6.99 (s, 2H), 6.93 (b, 1H), 6.44 (d,1H), 5.98 (b, 1H) 5.32 (d, 1H), 5.13 (d, 1H), 5.05 (m, 2H) 4.68 (m, 3H),4.48 (b, 1H), 4.36 (b, 1H), 4.24 (b, 1H), 3.71 (s, 3H), 3.68 (m, 1H),3.60 (b, 1H), 3.38 (b, 1H), 3.11 (b, 1H), 2.97 dd, 1H), 2.94 (m, 1H),2.65 (dd, 1H), 2.25–2.45 (m, 4H) 1.88–2.16 (m, 4H), 1.65 (m, 2H), 1.45(s, 9H), 1.41 (s, 9H).

Step 5E: Synthesis of tert-butyl(S,S,S)-4-amino-4-(N-(1-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoate

The product of 5D (58 mg, 57 μmol) was hydrogenated according to theprocedure of step 1I, to yield the product (44 mg, 88%) as a whitesolid, which was not further purified but was lyophilized in 0.1%aqueous trifluoroacetic acid/acetonitrile (1:1) and used as thetrifluoroacetate salt in the next step. LRMS (ES): 875.6 [M+H]⁺, 438.5[M+2H]⁺²,;

Step 5F: Synthesis of tert-butyl(S,S,S)-4-(N-(1-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoate

The product of 5E (24.4 mg, 20 μmol) was reacted with DOTAtri-tert-butyl ester as in step 1J, to afford the product (19.6 mg, 55%)as a trifluoroacetate salt after lyophilization.

LRMS (ES): 1430.0 [M+H]⁺, 715.7 [M+2H]⁺², 477.8 [M+3H]⁺³; HRMS(ESI):Calculated for C₇₃H₁₁₃N₁₂O₁₇—1429.8347, found—1429.838;

Step 5G: Synthesis of(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)butanoicacid

The product of 5F (13 mg, 7.4 μmol) was deprotected and purified as instep 1K, to afford the product (6.5 mg, 55%) as a trifluoroacetate saltafter lyophilization. LRMS (ES): 1135.6 [M+H]⁺, 568.5 [M+2H]⁺², 379.6[M+3H]⁺³; HRMS(ESI): Calculated for C₅₂H₇₁N₁₂O₁₇—1135.5060,found—1135.503;

Example 6 Preparation of(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoicacid

Step 6A: Synthesis of tert-butyl(S,S)-3-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate

The product of step 5D (65 mg, 130 μmol) was reacted withN-(carbobenzyloxy)-β-(tert-butyl)-□-(N-hydroxysuccinimidyl)aspartate (66mg, 156 μmol) and diisopropylethylamine (181 μL, 1.04 mmol) indimethylformamide (1.5 mL) with stirring at room temperature undernitrogen for 20 hr. The reaction was concentrated, and the residuedissolved in ethyl acetate. The organics were washed with water, 10%potassium hydrogen sulfate, water, and brine, and then concentrated. Theresidual oil was purified by flash chromatography on silica (EtOAc/MeOH,1%→10%) and the product fractions combined and evaporated to yield theproduct (76 mg, 73%) as an oil. LRMS (ES): 810.5 [M+H]⁺, 378.0; HRMS(ESI): Calculated for C₄₃H₅₂N₇O₉—810.3826, found—810.3819; ¹HNMR(600.1323 MHz, CDCl₃) 7.25–7.38 (m, 12H), 7.18 (m, 2H), 7.07 (b, 1H),6.99 (s, 2H), 6.39 (d, 1H), 6.18 (b, 1H) 5.30 (d, J=16.2 Hz, 1H), 5.09(m, 2H), 5.04 (m, 1H) 4.67 (m, 4H), 4.50 (b, 1H), 4.36 (b, 1H), 3.69 (s,3H), 3.62 (d, J=18.6 Hz, 1H), 3.45 (b, 1H), 3.14 (m, 1H), 2.94 (dd, 1H),2.86 (m, 2H), 2.62 (m, 2H), 1.60 (m, 2H), 1.39 (s, 9H).

Step 6B: Synthesis of tert-butyl(S,S)-3-amino-3-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)propanoate

The product of 6A (70 mg, 86 μmol) was hydrogenated according to theprocedure of step 1I, to yield the product (55 mg, 95%) as a whitesolid, which was not further purified but was lyophilized in 0.1%aqueous trifluoroacetic acid/acetonitrile (1:1) and used as thetrifluoroacetate salt in the next step. LRMS (ES): 676.5 [M+H]⁺, 339.0[M+2H]⁺², 310.9.

Step 6C: Synthesis of tert-butyl(S,S)-3-(N-(3-(3,6-diaza-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)propanoate

The product of 6B (22.4 mg, 22 μmol) was reacted with DOTAtri-tert-butyl ester and purified as in step 1J, to afford the product(16.6 mg, 44%) as a trifluoroacetate salt after lyophilization. LRMS(ES): 1230.9 [M+H]⁺, 616.2 [M+2H]⁺², 411.3 [M+3H]⁺³; HRMS(ESI):Calculated for C₆₃H₉₆N₁₁O₁₄—1230.7138, found—1230.715;

Step 6D: Synthesis of(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)propanoicacid

The product of 5F (14 mg, 8.3 μmol) was deprotected and purified as instep 1K, to afford the product (4.6 mg, 47%) as a trifluoroacetate saltafter lyophilization. LRMS (ES): 992.6 [M+H]⁺, 497.0 [M+2H]⁺², 331.8[M+3H]⁺³; HRMS(ESI): Calculated for C₄₆H₆₂N₁₁O₁₄—992.4478,found—992.4457;

Example 7 Synthesis of(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoicacid

Step 7A: Synthesis of tert-butyl(S,S,S,S,S,S)4-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-(N-(1-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

The product of step 1I (65 mg, 54.6 μmol) is dissolved in DMF (1 mL)along with HBTU (25 mg, 65 μmol), N-carbobenzyloxy-L-glutamic acid (7.3mg, 26 μmol), HOBT (7 mg, 52 μmol), and diisopropylethylamine (40 μL,225 μmol) under nitrogen. After stirring for 2 hrs, the reaction isconcentrated and purified by preparative HPLC (0.1% TFA/acetonitrilegradient, Zorbax C8, 21.5 mm×25 cm). The product may be obtained as thetrifluoroacetate salt after lyophilization.

Step 7B: Synthesis of tert-butyl(S,S,S,S,S,S)-4-(2-amino-4-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoylamino)-4-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoate

The product of step 7A is hydrogenated and isolated as in step 1I. Thismaterial is not further purified, but used directly in the followingstep.

Step 7C: Synthesis of tert-butyl(S,S,S,S,S,S,S,S)-4-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)oxycarbonyl)-1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

The product of step 7B is reacted as in step 5D to afford the product,which is purified by preparative HPLC.

Step 7D: Synthesis of tert-butyl(S,S,S,S,S,S,S,S)-4-amino-4-(N-(1-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)oxycarbonyl)-1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)butanoate

The product of step 7C is hydrogenated as in step 1I to afford theamine, which is not further purified but used directly in the next step.

Step 7E: Synthesis of tert-butyl(S,S,S,S,S,S,S,S)-4-(N-(1-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)oxycarbonyl)-1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl))propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoate

The product of step 7D is reacted with DOTA(OtBu)3-OH as in step 1J toafford the product as a solid after preparative HPLC purification andlyophilization. Alternatively, the product of 7B is reacted with theproduct of 7I in the presence of HBTU, HOBT, and diisopropylethylaminein dry dimethylformamide for 2 hours, after which the reaction isconcentrated and the residue purified by preparative HPLC to afford theproduct as a solid after lyophilization.

Step 7F: Synthesis of(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoicacid

The product of step 7D is deprotected as in step 1K to afford theproduct as a solid after preparative HPLC purification andlyophilization.

Step 7G: Synthesis of tert-butyl (S,S)-3,3-dimethyl-3-silabutyl2-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)pentane-1,5-dioate

The product of step 1G (1.25 g, 2.4 mmol) was reacted with2-trimethylsilylethanol (296 mg, 2.5 mmol) in the presence of ethyl[3-(N,N-dimethylaminopropyl]-carbodiimide hydrochloride (480 mg, 2.5mmol) and dimethylaminopyridine (250 mg, 1.2 mmol) in dimethylformamide(10 mL) at 0° C. The reaction was allowed to warm slowly to roomtemperature and stirred overnight. It was concentrated and the residuepartitioned between ethyl acetate and water. The aqueous layer wasextracted twice with ethyl acetate, and the combined organics washedwith water, 10% potassium hydrogen sulfate, and brine, and concentrated.The residue was purified by flash chromatography (ethyl acetate/hexane)to afford the product as an oil (1.1 g, 73%). LRMS (ES): 623.5 [M+H]⁺.

Step 7H: Synthesis of tert-butyl (S,S)-3,3-dimethyl-3-silabutyl2-(2-amino-4-((tert-butyl)oxycarbonyl)butanoylamino)pentane-1,5-dioate

The product of step 7G (1.09 g) was dissolved in 2-propanol (75 mL) with10% palladium on carbon (300 mg) and hydrogenated on a Parr shaker at 45psi for one hour. The reaction mixture was filtered on a bed of Celite,washed with 2-propanol, and concentrated to yield the product (803 mg,94%) as a clear oil. LRMS (ES): 489.5 [M+H]⁺, 977.7 [2M+H]⁺. ¹HNMR(600.1343 MHz, CDCl₃): 7.78 (m, 1H), 4.53 (m, 1H), 4.22 (m, 2H), 3.53(m, 1H), 1.80–2.41 (m, 10H), 1.43 (s, 18H), 1.01 (m, 2H), 0.02 (s, 9H).

Step 7I: Synthesis of tert-butyl (S,S)-3,3-dimethyl-3-silabutyl2-(4-((tert-butyl)oxycarbonyl)-2-(2-bromoacetylamino)butanoylamino)pentane-1,5-dioate

The product of step 7H (397 mg, 0.813 mmol) was dissolved in drytetrahydrofuran (5 mL) with diisopropylethylamine (180 μL, 1.05 mmol)and cooled to —10° C. under nitrogen. Bromoacetyl bromide (85 μL, 0.98mmol), dissolved in 10 mL tetrahydrofuran, was added dropwise to thecold solution, keeping T≦−5° C. The reaction was stirred in the cold for1.5 hr, and 25 μL methanol added. The solids were filtered and rinsedand the combined filtrate concentrated to a brown oil, which waspurified by flash chromatography (dichloromethane/ethyl acetate) toafford the product (388 mg, 78%) as a light tan oil. LRMS (ES):609.3/611.3 [M+H]⁺, 631.3/633.3 [M+Na]⁺, 185.3, 144.2. ¹HNMR (600.1343MHz, CDCl₃): 7.32 (m, 1H), 7.09 (m, 1H), 4.50 (m, 2H), 4.21 (m, 2H),3.87 (m, 2H), 2.31 (m, 2H), 2.13 (m, 2H), 1.99 (m, 2H), 1.97 (m, 2H),1.45 (s, 9H), 1.43 (s, 9H), 1.01 (m, 2H), 0.04 (s, 9H).

Step 7J: Synthesis of(S,S)-4-((tert-butyl)oxycarbonyl)-2-(4-((tert-butyl)oxycarbonyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoylamino)butanoicacid

The product of step 7H (214 mg, 0.416 mmol) was dissolved indimethylformamide (3 mL) and added to a solution of triethylamine (250μL) and DO3A tri-tert-butyl ester in dimethylformamide (3 mL). Thereaction was stirred for 4 days at room temperature, concentrated, andthe residue dissolved in ethyl acetate. This was washed with water andbrine, dried, and concentrated to an oil which was not further purifiedbut reacted directly with tetra-butylammonium fluoride (1.0M intetrahydrofuran, 1.25 mL) in tetrahydrofuran (2.5 mL). After stirringfor 2 hours, the reaction was treated with ether (50 mL) and water (50mL) and the layers separated. The aqueous layer was extracted with threeportions of ethyl acetate, and the combined organic layers concentratedto an oil. This was purified by preparative HPLC (0.1% trifluoroaceticacid/acetonitrile, Zorbax C-8, 21.5 mm×25 cm) and the product fractionslyophilized to afford 127 mg (32% for two steps) of the product as awhite solid. LRMS (ES): 943.3 [M+H]⁺, 887.2, 831.2, 775.5, 719.3, 663.2(loss of 1–5 tert-butyl) 444.3, 416.2, 388.3, 360.1, 332.1 [M-(1–5 tertbutyl) +2H]⁺². ¹HNMR (600.1343 MHz, CDCl₃): 9.05 (b, 1H), 8.2 (b, 4H)7.36 (b, 1H), 4.34 (m, 2H), 2.77–4.23 (very broad humps, 24H), 2.31 (m,4H), 2.13 (m, 2H), 1.93 (m, 2H), 1.47 (d, 18H), 1.43 (m, 27H).

Example 8 Synthesis of(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-Diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoicacid

Step 8A: Synthesis of ditert-butyl(S,S)-2-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)pentane-1,5-dioate

Gamma-tert-butyl-N-carbobenzyloxyglutamic acid N-hydroxy-succinimideester is dissolved in DMF with diisopropylethylamine.Bis(tert-butyl)glutamate hydrochloride is added and the reaction stirredfor one hour. The reaction is concentrated, water added, and the mixtureextracted with ethyl acetate. The combined organic layers are washedwith water, 10% potassium hydrogen sulfate, and brine, and thenconcentrated. The product is purified by flash chromatography.

Step 8B: Synthesis of tert-butyl methyl(S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)-4-((phenylmehtoxycarbonylamino)butanoylamino)pentane-1,5-dioate

The product of 8a is dissolved in one volume of dichloromethane andtreated with excess triethylsilane and one volume of trifluoroaceticacid. The reaction is stirred under nitrogen for three hours and thenconcentrated to an oil. The triacid residue is dissolved indimethylformamide and treated with excess gamma-tert-butyl-alpha-methylglutamate, HBTU, HOBT, and diisopropylethylamine with stirring undernitrogen for 4–5 hours. The reaction is concentrated, partitioned intowater/ethyl acetate and extracted with more ethyl acetate. The combinedorganics are washed with water and brine and concentrated to an oil,which is purified by flash chromatography using dichloromethane/ethylacetate/methanol.

Step 8C: Synthesis of methyl(S,S,S,S,S,S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-2-(4-(N-(1,3-bis(N-(3-(N-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)-4-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

The product of 8b is dissolved in one volume of dichloromethane andtreated with excess triethylsilane and one volume of trifluoroaceticacid. The reaction is stirred under nitrogen for three hours and thenconcentrated to an oil.

A threefold excess of the product of step 1F is treated in the samefashion with trifluoroacetic acid and triethylsilane and concentrated toan oil. The two residues are dissolved in dimethylformamide, combined,and treated with HBTU, HOBT, and diisopropylethylamine with stirringunder nitrogen, following disappearance of starting material by HPLC.When complete, the reaction is concentrated, partitioned intowater/ethyl acetate and extracted with more ethyl acetate. The combinedorganics are washed with water and brine and concentrated to an oil,which is purified by preparative HPLC using a 0.1% trifluoroaceticacid/acetonitrile gradient to afford the product as a powder afterlyophilization.

Step 8D: Synthesis of methyl(S,S,S,S,.S,S,S,S)-2-(4-amino-4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoate

The product of step 8C is dissolved in methanol with 10% palladium oncarbon and 2 equivalents of acetic acid in a Parr bottle. The mixture ishydrogenated at 55 psi in a Parr shaker, following by HPLC until all thestarting material has been reacted. The reaction is filtered throughCelite, concentrated, and the residual oil lyophilized fromwater/acetonitrile to yield the product as a powder, to be used directlyin the next step.

Step 8E: Conjugation of 8D with 7I

The product of step 8D is reacted with the product of step 7I asdescribed in the alternate synthesis of 7E to afford the product as asolid after preparative HPLC purification and lyophilization.

Step 8F: Synthesis of(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoicacid

The product of step 8E is dissolved in 2:1 methanol/tetrahydrofuran andexcess lithium hydroxide (3M solution) added. The solution is stirred,following by HPLC, until all the methyl esters have been hydrolyzed. Thereaction is quenched with solid citric acid, concentrated, andredissolved in one volume of dichloromethane. The solids are filteredand the filtrate treated with excess triethylsilane and one volume oftrifluoroacetic acid. The solution is stirred under nitrogen, followingby HPLC, until all of the tert-butyl esters have been hydrolyzed. Thereaction mixture is concentrated and directly purified by preparativeHPLC using 0.1% formic acid/acetonitrile gradient on a Zorbax C-8 columnto afford the product after lyophilization.

Example 9 Preparation of(S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

Step 9A: Synthesis ofN-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)(tert-butoxy)formamide

A solution of at least three equivalents of4,7,10-trioxa-1,13-tridecanediamine in tetrahydrofuran is cooled to 0°C., and a solution of one equivalent of di-tert-butyl dicarbonate inacetonitrile is added dropwise with stirring. The solution is stirredunder nitrogen overnight and then concentrated. The residue is dissolvedin ether and washed with five portions of saturated sodium chloride. Theorganic layer is dried over magnesium sulfate, filtered and concentratedto an oil, which is purified by flash chromatography to afford themonoamine.

Step 9B: Synthesis of tert-butyl3-(((3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)amino)methyl)-4-fluorobenzoate

The product of step 9A is treated with crudetert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate, as described in step1A, to afford the product after flash chromatography.

Step 9C: Synthesis of methyl(S)-3-(N-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate

The product of step 9B is treated with Z-aspartic acid-β-methyl ester asdescribed in step 1B, to afford the product after flash chromatography.

Step 9D: Synthesis of methyl(S)-3-amino-3-(N-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)carbamoyl)propanoate

The product of step 9C is treated as in step 1C, and used directly inthe following step.

Step 9E: Synthesis of methyl(S)-2-(2,5-diaza-9-((tert-butyl)oxycarbonyl-5-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of step 9D is treated as in step 1D, to afford the productafter flash chromatography.

Step 9F: Synthesis of(S)-2,5-diaza-5-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-3-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-9-carboxylicacid

The product of step 9E is treated as in step 1E, to afford the productafter flash chromatography.

Step 9G: Synthesis of methyl(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(3-(2-(2-(3-((tert-butoxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of step 9F is treated as in step 1F, to afford the productafter flash chromatography.

Step 9H: Synthesis of(S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

The product of step 9G is treated as in step 2G, and the isolatedresidue then directly treated as in step 2H to afford the product afterpreparative HPLC and lyophilization.

Example 10 Preparation of(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)hexanoylamino)butanoicacid

Step 10A: Synthesis of methyl(S)-2-(5-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of step 9G is treated with trifluoroacetic acid andtriethylsilane in dichloromethane for 30 minutes and the reaction thenconcentrated to an oil. Toluene is added and the solution reconcentratedto an′ oil, which is used directly in the next step.

Step 10B: Synthesis of(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)hexanoylamino)butanoicacid

The product of step 10A is treated in several steps as defined inexample 7, steps 7A–7F, substituting step 10A product for step 1Iproduct as a starting material in step 7A. The product is obtained as asolid after preparative HPLC purification and lyophilization.

Example 11 Synthesis of(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

Step 11A: Synthesis of tert-butyl methyl(S,S)-2-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)pentane-1,5-dioate

This process is carried out as in step 1G, except starting withalpha-methyl-gamma-tert-butylglutamate.

Step 11B: Synthesis of methyl(S,S)-4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoate

The product of step 11A is dissolved in dichloromethane, followed byaddition of trifluoroacetic acid (to form a 35% solution). This isstirred under nitrogen until the starting material and monoacid havedisappeared by HPLC, and then the solution is concentrated. The residueis dissolved in dimethylformamide along with 2.5 equivalents of1-amino-1-deoxysorbitol, 2.5 equivalents of HBTU, 2 equivalents ofhydroxybenzotriazole hydrate, and 3 equivalents diisopropylethylamine.The solution is stirred for two hours, concentrated, and the residuepurified by preparative HPLC.

Step 11C: Synthesis of(S,S)-4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)butanoicacid

The product of step 11B is dissolved in tetrahydrofuran/methanol (1:1)and treated with excess 3N aqueous lithium hydroxide. The reaction isfollowed by HPLC for disappearance of starting material. The reaction isconcentrated, diluted with additional water, and purified by passagedown an acidic ion exchange column. The product fractions arelyophilized to afford the product as a solid.

Step 11D: Synthesis of methyl(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(phenylmethoxy)carbonylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of step 2G is dissolved in dichloromethane and stirred withtrifluoroacetic acid and triethylsilane for 15 minutes. The solution isconcentrated, and the residue dissolved in dimethylformamide with theproduct of step 11C, HBTU, hydroxybenzotriazole hydrate, anddiisopropylethylamine. The reaction is stirred, following by HPLC fordisappearance of starting materials. When complete, the solution isconcentrated and the residue purified by preparative HPLC. The productsolutions are lyophilized to afford the product.

Step 11E: Synthesis of methyl(S,S,S)-2-(5-(6-(2-(2-amino-4(-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)butanoylamino)-4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)butanoylamino)hexyl)-2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate

The product of step 11D is treated as in step 1I, to afford the amineafter concentration.

Step 11F: Synthesis of(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

The product of step 11E is reacted as in step 1J to afford the productafter preparative HPLC purification.

Step 11G: Synthesis of(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid

The product of step 11F is treated as in step 1K, to afford the productafter preparative HPLC purification.

Example 12 Synthesis of(S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoicacid

Step 12A: Synthesis of H-Asp(OtBu)-D-Phe-Lys(Cbz)-Arg(Mtr)-Gly-OH

This peptide is prepared using an Advanced Chemtech Model 90 synthesizerusing standard Fmoc protocols. The starting resin is4-[4-hydroxymethyl)-3-methoxy-phenoxy]butanoyl benzhydrylamine resinpreloaded with Fmoc-glycine (Fmoc-Gly-HMPB-BHA). Synthesis of theprotected linear peptide is achieved through sequential coupling (for 3hrs) of the amino acidsN-alpha-Fmoc-N⁹-4-methoxy-2,3,6-trimethylbenzenesulfonyl-1-arginine,N-alpha-Fmoc-N-epsilon-benzyloxycarbonyl-L-lysine, Fmoc-phenylalanine,and Fmoc-gamma-tert-butyl aspartic acid, using HBTU and HOBT as couplingagents. The couplings are carried out with five equivalents of aminoacid, HBTU, HOBT, and diisopropylethylamine in dimethylformamide. Fmocdeprotections are accomplished with 20% piperidine in DMF for 30minutes. The protected linear peptide is cleaved from the resin with 1%trifluoroacetic acid in dichloromethane and the peptide solutioncollected in 10% pyridine in methanol. The crude peptide is obtained byconcentrating the solvents in vacuo and triturating with diethyl ether.The peptide is purified by preparative HPLC and the product fractionsare lyophilized.

Step 12B: Synthesis of cyclo{Lys(Cbz)-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}

HBTU (0.7 mmol) and hydroxybenzotriazole (0.5 mmol) are dissolved indimethylformamide (10 mL). The solution is warmed to 60° C. undernitrogen and a solution of the product of step 12 A (0.4 g) anddiisopropylethylamine (1.5 mmol) in dimethylformamide (10 mL) addedslowly. The solution is stirred at this temperature for 4 hours undernitrogen. The solution is concentrated and the residue triturated withethyl acetate. The resulting solids are washed with ethyl acetate anddried under vacuum to afford the product, which is used directly in thenext step.

Step 12C: Synthesis of cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}

The product of step 12 B is dissolved in 2-propanol and 10% palladium oncarbon added with stirring. Hydrogen gas is gently bubbled into thereaction mixture until all of the starting material is consumed by HPLCanalysis. The reaction mixture is filtered through a bed of Celite andthe filtrate concentrated. The residue is not further purified but useddirectly in the following step.

Step 12D: Synthesis of tert-butyl(S,S)-4-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-(((phenylmethoxy)carbonylamino)butanoate

The product of step 2F is dissolved in dichloromethane andtrifluoroacetic acid added (30% solution). The reaction is stirred 30minutes and concentrated. The residue is dissolved in dimethylformamideandN-carbobenzyloxy-gamma-tert-butyl-alpha-N-hydroxysuccinimidylglutamateadded, along with excess diisopropylethylamine. The reaction is stirredfor four hours and concentrated. The residue is purified by preparativeHPLC and the fractions lyophilized to afford the product as a solid.

Step 12E: Synthesis of(S,S)-4-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-(((phenylmethoxy)carbonylamino)butanoyl-cyclo{Lys-Arg(Mtr)-Gly-Asp(ToBu)-D-Phe}conjugate

The product of step 12D is dissolved in one volume of dichloromethane,followed by one volume of trifluoroacetic acid and 5 equivalents oftriethylsilane. The solution is stirred for four hours and concentrated.The residue is dissolved in dimethylformamide containing the product ofstep 12C, HBTU, and hydroxybenzotriazole hydrate. Diisopropylethylamineis added to this mixture with stirring under nitrogen, following by HPLCfor disappearance of the starting materials. When complete, the reactionis concentrated and the residue purified by preparative HPLC. Theproduct fractions are combined and lyophilized.

Step 12F: Synthesis of(S,S)-4-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-amino)butanoyl)-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}conjugate

The product of step 12E is treated as in step 8D. The product is notfurther purified, but used directly in the next step.

Step 12G: Synthesis of tert-butyl(S,S,S,S)-4-(N-(1-N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}carbamoyl)propyl)carbamoyl-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclcododecyl)acetylamino)butanoate

The product of step 12F is treated as in step 8E to afford the productafter preparative HPLC purification.

Step 12H: Synthesis of(S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclcododecyl)acetylamino)butanoicacid

The product of step 12G is dissolved in tetrahydrofuran and excesslithium hydroxide added as a 3N solution in water. The solution isstirred under nitrogen, following by HPLC for disappearance of startingmaterial. When this is complete, the reaction is acidified with 10%potassium hydrogen sulfate and concentrated. The residue is dissolved inneat trifluoroacetic acid containing thioanisole and stirred at roomtemperature under nitrogen, following the multiple deprotections byHPLC, until complete. The reaction is concentrated and the crude residuepurified by preparative HPLC.

The following procedure describe the synthesis of radiopharmaceuticalsof the present invention of the formula^(99m)Tc(VnA)(tricine)(phosphine), in which (VnA) represents avitronectin receptor antagonist compound of the present invention bondedto the Tc through a diazenido (—N═N—) or hydrazido (═N—NH—) moiety. Thediazenido or hydrazido moiety results from the reaction of thehydrazinonicotinamido group, present either as the free hydrazine orprotected as a hydrazone, with the Tc-99m. The other two ligands in theTc coordination sphere are tricine and a phosphine.

Examples 13–14 Synthesis of Complexes [^(99m)Tc (HYNIC-VnA) (tricine)(TPPTS)]

To a lyophilized vial containing 4.84 mg TPPTS, 6.3 mg tricine, 40 mgmannitol, succinic acid buffer, pH 4.8, and 0.1% Pluronic F-64surfactant, was added 1.1 mL sterile water for injection, 0.2 mL (20 μg)of the appropriate HYNIC-conjugated vitronectin antagonist (VnA) indeionized water or 50% aqueous ethanol, and 0.2 mL of ^(99m)TcO₄ ⁻ (50±5mCi) in saline. The reconstituted kit was heated in a 100° C. water bathfor 15 minutes, and was allowed to cool 10 minutes at room temperature.A sample of the reaction mixture was analyzed by HPLC. The RCP resultsare listed in the table 1.

TABLE 1 Analytical and Yield Data for ^(99m)Tc(VnA) (tricine) (TPPTS)Complexes Example Reagent Ret. Time % No. No. (min) Yield 13 2 8.9* 8614 3 22.5** 46 *The HPLC method using a reverse phase C₁₈ Zorbax column(4.6 mm × 25 cm, 80 Å pore size) at a flow rate of 1.0 mL/min with agradient mobile phase from 100% A (25 mM pH 8.0 phosphate buffer) to 20%B (acetonitrile) at 20 min. **The HPLC method using a reverse phase C₁₈Zorbax column (4.6 mm × 25 cm, 80 Å pore size) at a flow rate of 1.0mL/min with a gradient mobile phase from 100% A (10 mM pH 6.0 phosphatebuffer) to 25% B (acetonitrile) at 40 min.

Examples 15–22 Synthesis of ¹⁷⁷Lu and ⁹⁰Y Complexes

To a clean sealed 10 mL vial was added 0.5 mL of a solution of theappropriate conjugate (200 μg/mL in 0.25 M ammonium acetate buffer, pH7.0), followed by 0.05–0.1 mL of gentisic acid (sodium salt, 10 mg/mL in0.25 M ammonium acetate buffer, pH 7.0) solution, 0.3 mL of 0.25 Mammonium acetate buffer (pH 7.0), and 0.05 mL of ¹⁷⁷LuCl₃ solution (˜200mCi/mL) or ⁹⁰YCl₃ solution (100–200 mCi/mL) in 0.05 N HCl. The resultingmixture was heated at 100° C. for 35 min. After cooling to roomtemperature, a sample of the resulting solution was analyzed byradio-HPLC and ITLC. For ⁹⁰Y complexes, the sample has to be diluted15–20 fold before the radio-HPLC analysis. The ITLC method used GSsilica-gel paper strips and a 1:1 mixture of acetone and saline aseluant. The analytical and yield data are shown in Table 2.

TABLE 2 Analytical and Yield Data for Lu-177 and Y-90 Complexes ExampleReagent Ret. Time No. Ex. No. Isotope (min) % RCP 15 1 ¹⁷⁷Lu 14.1 94 161 ⁹⁰Y 14.0 92 17 1 ¹⁴⁹Pm 14.0 94 18 5 ¹⁷⁷Lu 14.1 94 19 5 ⁹⁰Y 14.7 93 205 ¹⁴⁹Pm 15.0 94 21 6 ¹⁷⁷Lu 17.1 94 22 6 ⁹⁰Y 17.4 84

HPLC Method

-   Column: Zorbax C18, 25 cm×4.6 mm-   Flow rate: 1.0 mL/min-   Solvent A: 25 mM sodium phosphate buffer, pH 6.0-   Solvent B: 100% CH₃CN

Gradient I t (min) 0 20 21 30 31 40 % Solvent B 0 20 60 60 0 0

The identity of the Lu-177 complexes of Examples 15, 18, 21 were furtherconfirmed by LC-MS. The MS data are shown in Table 3.

TABLE 3 Mass Spec. Data for Lu-177 Complexes Example Atomic No. FormulaWeight M + H⁺ 15 C₅₀H₆₅LuN₁₂O₁₇ 1280.4 1282.0 18 C₅₂H₆₇LuN₁₂O₁₇ 1306.41307.3 21 C₄₆H₅₈LuN₁₁O₁₇ 1163.4 1164.2

Example 23 Synthesis of the ¹¹¹In Complex of the Conjugate of Example 1

To a lead shielded and closed autosampler vial was added 65 μg of theconjugate of Example 1 and 1.5 mg gentisic acid, sodium salt dissolvedin 65 μL ammonium acetate buffer (0.4 M, pH 4.7) followed by theaddition of 1.8 mCi, 15 μL In-111 in 0.05 N HCl (specific activity: 36μg/mCi). The reaction mixture was heated at 70–80° C. for 60 min andanalyzed by HPLC and ITLC. The radiolabeling yield was 91% and theretention time was 9.8 min.

HPLC Method

-   Column: Zorbax C18, 25 cm×4.6 mm-   Flow rate: 1.0 mL/min-   Solvent A: 10 mM sodium phosphate buffer, pH 6.0-   Solvent B: 100% CH₃CN

Gradient I t (min) 0 20 21 30 31 40 % Solvent B 5 20 60 60 5 5The ITLC method used GS silica-gel paper strips and a 1:1 mixture ofacetone and saline as eluant.

Examples 24–25 Synthesis of the ¹¹¹In Complex of the Conjugates ofExample 5 and 6

To a lead shielded and closed autosampler vial was added 100 μg of theappropriate conjugate of the present invention dissolved in 100 μLammonium acetate buffer (0.2 M, pH 4.7) followed by 2.3 mCi, 25 μLIn-111 in 0.05 N HCl. The solutions were heated at 100° C. for 30 minand analyzed by HPLC and ITLC. The radiolabeling yield for Example 24was 76% and the retention time was 9.4 min. The radiolabeling yield forExample 25 was 87% and the retention time was 17.2 min.

The ITLC method used GS silica-gel paper strips and a 1:1 mixture ofacetone and saline as eluant.

HPLC Method (Example 24)

-   Column: Zorbax C18, 25 cm×4.6 mm-   Flow rate: 1.0 mL/min-   Solvent A: 10 mM sodium phosphate buffer, pH 6.0-   Solvent B: 100% CH₃CN

Gradient I t (min) 0 20 21 30 31 40 % Solvent B 5 20 60 60 5 5

HPLC Method (Example 25)

-   Column: Zorbax C18, 25 cm×4.6 mm-   Flow rate: 1.0 mL/min-   Solvent A: 0.1% TFA in water-   Solvent B: 100% CH₃CN

Gradient I t (min) 0 20 21 30 31 40 % Solvent B 5 20 60 60 5 5

Example 26 Preparation of sodium1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid-dodecoanoate conjugate

Step 26A: Synthesis of sodium1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid-dodecoanoate conjugate

1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine monosodium salt (DPPE)(1.25 g, 0.5 mmol) is dissolved under nitrogen in chloroform (15 mL)along with disuccinimidyl dodecanoate (0.212 g, 0.5 mmol and the productof step 4A (367 mg, 0.5 mmol). They are stirred for 5 minutes, whensodium carbonate (0.5 mmol) and sodium sulfate (0.5 mmol) is added. Thereaction is stirred 18 hrs, filtered, and concentrated. The residue ispurified to obtain the title compound.

Step 26B: Preparation of Contrast Agent Composition

The product of step 13A is admixed with three other lipids,1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline, andN-(methoxypolyethylene glycol5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine inrelative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous solutionof this lipid admixture (1 mg/mL), sodium chloride (7 mg/mL), glycerin(0.1 mg/mL), and propylene glycol (0.1 mL/mL) at pH 6–7 is then preparedin a 2 cc glass vial. The air in the vial is evacuated and replaced withperfluoropropane and the vial is sealed. The ultrasound contrast agentcomposition is completed by agitating the sealed vial in a dentalamalgamator for 30–45 seconds to form a milky white solution.

Example 27 Preparation ofDPPE-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]-dodecoanoate conjugate

Step 27A: Synthesis ofω-amino-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]

A solution of N-Boc-ω-amino-PEG₃₄₀₀-succinimidyl ester (1 mmol) and theproduct of step 4A (1 mmol) in DMF (15 mL) is treated withdiisopropylethylamine (3 mmol) and stirred under nitrogen for 18 hr. Thesolution is concentrated and the residue dissolved in dichloromethane (8mL) to which trifluoroacetic acid (6 mL) is added. The solution isstirred for 30 minutes, and then concentrated under vacuum. The productis isolated by trituration with diethyl ether.

Step 27B: Synthesis ofDPPE-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]-dodecoanoate conjugate

A solution of disuccinimidyl dodecanoate (0.5 mmol), DPPE (0.5 mmol),and the product of step 14A (0.5 mmol) are added to 10 mL chloroformwith stirring under nitrogen. Sodium carbonate (1 mmol) and sodiumsulfate (1 mmol) are added and the solution is stirred at roomtemperature for 18 hrs. The reaction is filtered, the solventconcentrated, and the residue purified to obtain the title compound.

Step 27C: Preparation of Contrast Agent Composition

The product of step 14B is admixed with three other lipids,1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline, andN-(methoxypolyethylene glycol5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine inrelative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous solutionof this lipid admixture (1 mg/mL), sodium chloride (7 mg/mL), glycerin(0.1 mg/mL), and propylene glycol (0.1 mL/mL) at pH 6–7 is then preparedin a 2 cc glass vial. The air in the vial is evacuated and replaced withperfluoropropane and the vial is sealed. The ultrasound contrast agentcomposition is completed by agitating the sealed vial in a dentalamalgamator for 30–45 seconds to form a milky white solution.

Example 28 Preparation of[(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)carbamoyl)propyl)carbamoyl]-□-amino-PEG₃₄₀₀-dodecanoate-DPPEconjugate

Step 28A: Synthesis of[(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)carbamoyl)propyl)carbamoyl]-ω-amino-PEG₃₄₀₀

The product of step 4B (1 mmol) was deprotected as described in step 4Cand added to a solution of N-Boc-(β-amino-PEG₃₄₀₀-succinimidyl ester (1mmol) in DMF (15 mL). Diisopropylethylamine (3 mmol) is added and thesolution stirred under nitrogen for 18 hr. The solution is concentratedand the residue dissolved in dichloromethane (8 mL) to whichtrifluoroacetic acid (6 mL) is added. The solution is stirred for 30minutes, and then concentrated under vacuum. The product is isolated bytrituration with diethyl ether.

Step 28B: Synthesis ofDPPE-PEG₃₄₀₀-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)aceticacid]-dodecoanoate conjugate

A solution of disuccinimidyl dodecanoate (0.5 mmol), DPPE (0.5 mmol),and the product of step 15A (0.5 mmol) are added to 10 mL chloroformwith stirring under nitrogen. Sodium carbonate (1 mmol) and sodiumsulfate (1 mmol) are added and the solution is stirred at roomtemperature for 18 hrs. The reaction is filtered, the solventconcentrated, and the residue purified to obtain the title compound.

Step 28C: Preparation of Contrast Agent Composition

The product of step 15B is admixed with three other lipids,1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline, andN-(methoxypolyethylene glycol5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine inrelative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous solutionof this lipid admixture (1 mg/mL), sodium chloride (7 mg/mL), glycerin(0.1 mg/mL), and propylene glycol (0.1 mL/mL) at pH 6–7 is then preparedin a 2 cc glass vial. The air in the vial is evacuated and replaced withperfluoropropane and the vial is sealed. The ultrasound contrast agentcomposition is completed by agitating the sealed vial in a dentalamalgamator for 30–45 seconds to form a milky white solution.

Example 29 Synthesis of4-[N-(3-{(2R)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl](4S)-4-[(4S)-4-(N-{(1S)-1-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-3-carboxypropyl}carbamoyl)-4-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}butanoylamino]butanoicacid

Step 29A: Synthesis of

The product of step 1F (100 mg, 0.172 mmol) was dissolved indichloromethane (4 mL) and treated with trifluoroacetic acid (4 mL) andtriethylsilane (160 uL) under nitrogen. The reaction was stirred for 25minutes and concentrated under vacuum, treated with toluene (5 mL) andreconcentrated. The residue was dissolved in DMF (2 mL) and treated withtert-butyl 2,5-dioxopyrrolidinyl(2S)-2-](phenylmethoxy)carbonyl-amino]pentane-1,5-dioate (85 mg, 0.19mmol) and diisopropylethylamine (135 uL, 0.775 mmol). The mixture wasstirred under nitrogen for 1 hour and then partitioned into ethylacetate/water (1:1, 100 mL). The layers were separated and the aqueouslayer extracted with two more portions of ethyl acetate. The combinedorganic layer was washed with water and brine, dried over sodiumsulfate, filtered, and concentrated to afford the product as a pale oilwhich solidified under vacuum (145 mg, 105%). This was used directly inthe next step. LRMS (ES): 798.4 [M+H]⁺, 100%

Step 29B: Synthesis of

The product of step 29A is deprotected as in step 6B to afford an impureoil. This was purified by preparative HPLC (Vydac C18, 2.25×25 cm, 90%acetonitrile/water/0.1% TFA; 5–55% B over 25 minutes), the productfractions combined, frozen, and lyophilized to afford the product as thebis-TFA salt (100 mg, 97%). LRMS (ES): 664.4 ([M+H]+, 20%), 333.0([M+2H]+2, 100%).

Step 29C: Synthesis ofbis-2,3,5,6-tetrafluorophenyl(2S)-2-[(tert-butoxy)carbonylamino]pentane-1,5-dioate

Boc-Glutamic acid (4.0 g, 16.2 mmol) was dissolved in DMF (60 mL) with2,3,5,6-tetrafluorophenol (6.5 g, 39 mmol). To this was added(3-dimethylaminopropyl)ethyl carbodiimide hydrochloride (7.4 g, 39 mmol)and the solution was stirred 18 hr. The reaction was concentrated andthe residue partitioned between ethyl acetate and water. The aqueouslayer was extracted three times with ethyl acetate, and the combinedorganic layer was washed with 0.1N HCl, water, and brine. It wasconcentrated to a white solid which was washed with two portions ofacetonitrile and dried under vacuum to afford the product as a whitesolid (6.2 g, 70%) with mp=123.5–124.5C. LRMS: 566.0 [M+Na]⁺. ¹HNMR(600.1343 MHz, CDCl₃): 7.02 (m, 2H), 5.14 (m, 1H), 4.80 (m, 1H), 2.92(m, 2H), 2.53 (m, 1H), 2.80 (m, 1H), 1.47 (s, 9H).

Step 29D: Synthesis of

The product of step 29B (95 mg, 94 umol) was treated with the product of29C (24.4 mg, 45 umol) and diisopropylethylamine (99 μL, 570 umol) inDMF and allowed to stir under nitrogen for 20 hr. The reaction wasconcentrated, water added and extracted three times with ethyl acetate.The combined organics were washed with 0.1N NaOH, water, and brine,dried over magnesium sulfate, filtered and concentrated to a white film(63 mg, 91%) which was not further purified but used directly in thenext step. LRMS (ES): 1538.1 ([M+H]⁺, 5%), 770.0 [M+2H]⁺², 100%), 514.0([M+3H]⁺³, 25%).

Step 29E: Synthesis of

The product of step 29D (60 mg, 39 umol) was dissolved indichloromethane (2.5 mL) under nitrogen. Trifluoroacetic acid (2.5 mL)and triethylsilane were added (100 μL) and the solution stirred for 1.5hr. The reaction was concentrated and chased with toluene (2×5 mL). Theresidue was dissolved in THF/methanol (1:1, 3 mL) and treated with a 3Nsolution of lithium hydroxide in water (260 uL, 390 umol). Afterstirring for 12 hours, another aliquot of lithium hydroxide (130 uL) wasadded and stirring continued for five hours. The reaction was acidifiedwith 0.1N HCl to pH=2 and concentrated. Purification by preparative HPLC(Vydac C18, 2.25×25 cm, 90% acetonitrile/water/0.1% TFA; 5–35% B over 50minutes), combining product fractions, and lyophilizing afforded theproduct as a white solid (23 mg 45%). LRMS (ES): 1298.4 ([M+H]⁺, 10%),649.9 ([M+2H]⁺², 30%), 433.6 ([M+3H]⁺³, 100%).

Step 29F: Synthesis of

The product of step 29E (20 mg, 14.1 umol) was dissolved in dry DMF (0.5mL) with diisopropylethylamine (15 μL, 85 umol) under nitrogen. Inanother flask under nitrogen, DOTA(OtBu)₃—OH (17 mg, 21 umol) wasdissolved in DMF with diisopropylethylamine (15 μL, 85 umol) and HBTU(6.7 mg, 18 umol) and stirred 10 minutes. The activated DOTA solution isadded in one portion to the amine and stirred for 30 minutes. Thereaction was concentrated and purified by preparative HPLC (Vydac C18,2.25×25 cm, 90% acetonitrile/water/0.1% TFA; 15–535% B over 50 minutes),combining product fractions, and lyophilizing afforded the product as awhite solid (8 mg, 30%). LRMS (ES): 1853.0 [M+H]⁺,

Step 29G: Synthesis of

The product of step 29F (7 mg) was dissolved in trifluoroacetic acid (2mL) with triethylsilane (200 μL) under nitrogen and stirred for 30minutes. The solution was concentrated and purified by prep HPLC (VydacC18, 2.25×25 cm, 50% acetonitrile/water/0.1% formic acid; 15–35% B over50 minutes). The product fractions were combined and lyophilized toafford a white solid (2 mg). LRMS (ES): 1684.6 ([M+H]⁺, 5%), 843.0([M+2H]⁺², 50%), 562.5 ([M+3H]⁺³, 100%).

Example 30 Synthesis of2-(4-{3-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]propyl}(2S)-7-{N-[2-(amidinoamino)ethyl]-N-methylcarbamoyl}-3-oxo-1H,2H,5H-benzo[f]1,4-diazepin-2-yl)aceticacid

Step 30A: Synthesis of

The product from step 1E (100 mg, 220 umol),N-[2-(methylamino)ethyl](phenylmethoxy)carboxamide hydrochloride (57 mg,230 umol), (3-dimethylaminopropyl)ethyl carbodiimide hydrochloride (51mg, 264 umol), and HOBT (31.2 mg, 230 umol) were dissolved in DMF (2.2mL) under nitrogen and the solution was stirred 18 hr. The reaction wasconcentrated and the residue partitioned between ethyl acetate andwater. The aqueous layer was extracted three times with ethyl acetate,and the combined organic layer was washed with 0.1N HCl, water, andbrine. It was dried over sodium sulfate, filtered, and concentrated to aclear oil, which was purified by flash chromatography (2% methanol/ethylacetate). Product fractions were combined and concentrated to yield theproduct as an oil (110 mg, 80%). LRMS (ES): 626.4 ([M+H]⁺, 100%), 648.4([M+Na]⁺, 100%) 1273.7 ([2M+Na]⁺, 15%).

Step 30B: Synthesis of

The product of Step 30A (110 mg) was treated as in step 1I to afford theproduct (98 mg, 100%) as a white solid. LRMS (ES): 492.4 ([M+H]⁺, 100%),514.4 ([M+Na]⁺, 30%)

Step 30C: Synthesis of

The product of step 30B (45 mg, 92 umol) was dissolved in DMF (0.6 mL)with diisopropylethylamine (33 μL, 185 umol), andtert-butyl-2-aza-3-[(tert-butoxy)carbonylamino]-3-methylthioprop-2-enoate(26.6 mg, 92 umol). Mercuric chloride (25 mg, 92 umol) was added and thereaction stirred 75 min. It was then diluted with ethyl acetate,filtered through Celite, and the solids rinsed. The combined filtratewas washed with water and brine, dried over sodium sulfate, filtered andconcentrated to afford a crude oil, which was purified by prep HPLC(Vydac C18, 2.25×25 cm, 90% acetonitrile/water/0.1% TFA; 10–70% B over30 minutes). The product fractions were combined and lyophilized toafford the product as a white solid (18 mg, 30%) which as a mixture ofproduct and deprotected material, which was used directly in the nextreaction. LRMS (ES): 734.4 [M+H]⁺, 634.4 [M-Boc+H]⁺.

Step 30D: Synthesis of

The product of step 30C (16 mg, 22 umol) was treated as in step 16E, andpurified by prep HPLC (Vydac C18, 2.25×25 cm, 90%acetonitrile/water/0.1% TFA; 0–25% B over 30 minutes). The productfractions were combined and lyophilized to afford the product as a whitesolid (6 mg, 52%). LRMS (ES): 420.2 ([M+H]⁺, 30%) 210.7 ([M+2H]⁺²,100%).

Step 30E: Synthesis of

The product of step 30D is treated as in step 3E, purified by prep HPLCand lyophilized to afford the product.

Example 31 Synthesis of2-[9-(N-{6-[(6-{[(1E)-1-aza-2-(2-sulfophenyl)vinyl]amino}(3-pyridyl))carbonylamino]hexyl}-N-(benzimidazol-2-ylmethyl)carbamoyl)(5S)-5,6,11-trihydro-dibenzo[b,e][7]annulen-5-yl]aceticacid

Step 31A: Synthesis of

The product of step 3A (300 mg, 0.66 mmol) and6-[(ethoxycarbonyl)methyl]-5,6,11-trihydrodibenzo[a,d][7]annulene-2-carboxylicacid (215 mg, 0.66 mmol, prepared according to W. H. Miller et al.,Bioorg. Med. Chem. Lett., 9(1999) 1807–1812) are treated as in step 3Bto yield the product after flash chromatography.

Step 31B: Synthesis of

The product of step 31A (100 mg, 0.15 mmol) is dissolved THF (3 mL) withlithium hydroxide (3N solution in water, 0.5 mL, 1.5 mmol) and stirred,monitoring for disappearance of starting material by HPLC. When thereaction is complete, the solution is acidified to pH=2 with 0.1N HCland the resulting solids are filtered and dried under vacuum to affordthe product, which is used directly in the following step.

Step 31C: Synthesis of

The product of step 31B is treated as in step 3C to afford the productas a solid after lyophilization.

Step 31D: Synthesis of

The product of step 31C is treated as in step 3E to afford the productas a yellow solid after prep HPLC purification and lyophilization.

Example 32 Synthesis of(2S)-2-[(2S)-4-(N-{(1S)-3-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-carboxypropyl}carbamoyl)-2-[(2S)-2-((2S)-4-carboxy-2-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}butanoylamino)-4-carboxybutanoylamino]butanoylamino]-4-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]butanoicacid

Step 32A: Synthesis of tert-butyl methyl(2S)-2-[(2S)-4-(N-{(1S)-3-[(tert-butyl)oxycarbonyl]-1-(methoxycarbonyl)propyl}carbamoyl)-2-[(phenylmethoxy)carbonylamino]butanoylamino]pentane-1,5-dioate

Cbz-glutamic acid (1 g, 3.56 mmol) was dissolved in DMF (20 mL) alongwith H-Glu(OtBu)OMe-HCl (1.9 g, 7.5 mmol), HBTU (3.4 g, 8.9 mmol), HOBT(1.01 g, 7.5 mmol), and diisopropylethylamine (2.2 mL, 12.5 mmol) undernitrogen. The reaction was stirred for 18 hours, concentrated, andpartitioned between water and ethyl acetate. The solids were filteredand the filtrate layers separated. The aqueous layer was extracted withethyl acetate and the combined organic layers washed with 10% sodiumcarbonate, water, 10% potassium hydrogen sulfate, water, and brine. Thesolution was dried over sodium sulfate, filtered, and concentrated toafford a golden oil which was purified by flash chromatography (4:1dichloromethane/ethyl acetate). The product fractions were combined andconcentrated to afford the product as a clear oil (1.3 g, 54%) whichsolidified under vacuum. LRMS (ES): 680.5 ([M+H]⁺, 100%), 702.5([M+Na]⁺, 20%)

Step 32B: Synthesis of methyl(2S)-2-[(2S)-4-(N-{(1S)-3-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-[(methoxycarbonyl)methyl]-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-(methoxycarbonyl)propyl}carbamoyl)-2-[(phenylmethoxy)carbonylamino]butanoylamino]-4-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-[(methoxycarbonyl)methyl]-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]butanoate

The product of step 1F (104 mg, 180 umol) was dissolved indichloromethane (2 mL) and trifluoroacetic acid (1 mL) added withstirring under nitrogen. The solution was stirred for 1 hour,concentrated under vacuum, and reconcentrated twice with toluene toafford the benzodiazepine amine as an oil which was used directly below.

The product of step 32A (43 mg, 63 umol) was dissolved indichloromethane (0.5 mL) and trifluoroacetic acid (0.9 mL) added withstirring under nitrogen. The solution was stirred for 2 hours,concentrated under vacuum, and reconcentrated twice with toluene toafford the dicarboxylic acid as an oil which was used directly below.

Both of these products were dissolved in DMF (1.5 mL) under nitrogen,and HBTU (60 mg, 150 umol), HOBT (20 mg, 140 umol), anddiisopropylethylamine (180 μL, 1.1 mmol) added. The solution was stirredfor 18 hours, concentrated, and the residue purified by preparative HPLC(Vydac C18, 2.12×25 cm, 90% acetonitrile/water/0.1% TFA; 10–55% B over25 minutes). The product fractions were combined and lyophilized toafford the product as a white solid (84 mg, 69%). LRMS (ES): 1488.7([M+H]⁺, 10%), 745.1 ([M+2H]⁺², 100%), 497.3 ([M+3H]⁺³, 100%)

Step 32C: Synthesis of tert-butyl(4S)-4-[N-((1S)-1-{N-[(1S)-1,3-bis(N-{(1S)-3-[N-(3-{(2S)-7-[N-(Benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-[(methoxycarbonyl)methyl]-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-(methoxycarbonyl)propyl}carbamoyl)propyl]carbamoyl}-3-[(tert-butyl)oxycarbonyl]propyl)carbamoyl]-4-[(phenylmethoxy)carbonylamino]butanoate

The product of step 32B (70 mg, 47 umol) was dissolved in methanol (5mL) and added to 10% palladium on carbon (40 mg) suspended in methanol(5 mL) under nitrogen in a pressure bottle. The slurry was hydrogenatedat 55 psi on a Parr apparatus for two hours, additional catalyst (35 mg)added, and repressurized. The hydrogenation was continued for anadditional 3 hours, at which time the reaction was filtered throughCelite, rinsed with methanol, and the combined filtrate concentrated toa clear oil (49 mg). This was dissolved in dry DMF (1.5 mL), along withthe product of step 1G (22 mg, 42 umol), HBTU (18 mg, 46 umol), HOBT(6.5 mg, 42 umol), and diisopropylethylamine (9 uL, 52 umol) in aflame-dried flask under nitrogen. The reaction was stirred for 5.5hours, concentrated, and the residue purified by preparative HPLC (VydacC18, 2.12×25 cm, 90% acetonitrile/water/0.1% TFA; 10–70% B over 30minutes). The product fractions were combined and lyophilized to affordthe product as a white solid (32 mg, 48%). LRMS (ES): 1859.2 ([M+H]⁺,5%), 930.1 ([M+2H]⁺², 85%), 620.8 ([M+3H]⁺³, 100%)

Step 32D: Synthesis of tert-butyl(4S)-4-[N-((1S)-1-{N-[(1S)-1,3-bis(N-{(1S)-3-[N-(3-{(2S)-7-[N-(Benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-[(methoxycarbonyl)methyl]-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-(methoxycarbonyl)propyl}carbamoyl)propyl]carbamoyl}-3-[(tert-butyl)oxycarbonyl]propyl)carbamoyl]-4-[2-(1,4,7,10-tetraaza-4,7,10-tris{[(tert-butyl)oxycarbonyl]methyl}cyclododecyl)acetylamino]butanoate

The product of step 32C (30 mg, 13.6 umol) was dissolved in methanol (6mL) and added to 10% palladium on carbon (45 mg) in methanol (6 mL) andacetic acid (120 uL). The mixture was hydrogenated for 6.5 hours at 55psi, filtered, concentrated, and the residue dissolved in 50%water/acetonitrile (20 mL), frozen, and lyophilized to yield a whitepowder (20.6 mg). This was dissolved in dry DMF (1 mL) along with HBTU(20 mg, 53 umol), HOBT (2.3 mg, 15 umol), and diisopropylethylamine (15uL, 75 umol). The reaction was stirred for 1.5 hours, concentrated, andthe residue purified by preparative HPLC (Vydac C18, 2.12×25 cm, 90%acetonitrile/water/0.1% TFA; 50–75% B over 26 minutes). The productfractions were combined and lyophilized to afford the product as a whitesolid (9.6, 30% LRMS (ES): 2279.5 ([M+H]⁺, 10%), 1140.3 ([M+2H]⁺², 20%),760.8 ([M+3H]⁺³, 100%). HRMS: Calculated for C₁₁₃H₁₆₄N₂₁O₂₉—2279.004;Found—2279.198.

Step 32E: Synthesis of(2S)-2-[(2S)-4-(N-{(1S)-3-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]-1-carboxypropyl}carbamoyl)-2-[(2S)-2-((2S)-4-carboxy-2-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}butanoylamino)-4-carboxybutanoylamino]butanoylamino]-4-[N-(3-{(2S)-7-[N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl]-2-(carboxymethyl)-3-oxo(1H,2H,5H-benzo[f]1,4-diazepin-4-yl)}propyl)carbamoyl]butanoicacid

The product of step 32D (8 mg, 3 umol) was dissolved in methanol/THF(1:1, 600 uL) and lithium hydroxide (3N solution, 10 uL, 30 umol) added.Additional aliquots of lithium hydroxide were added (20 uL at 1 hr, 3hr, and 5 hr) and the reaction worked up at 6 hours. It was acidifiedwith trifluoroacetic acid, concentrated, and the residue dissolved indichloromethane (0.6 mL) along with trifluoroacetic acid (0.8 mL) andtriethylsilane (100 uL). The solution was stirred under nitrogen for 20hours, concentrated, and the residue purified by preparative HPLC (VydacC18, 2.12×25 cm, 90% acetonitrile/water/0.1% TFA; 12–23% B over 50minutes). The product fraction was lyophilized to afford the product asa white solid (2.1 mg, 38%). LRMS (ES): 1942.6 ([M+H]⁺, 5%), 971.9([M+2H]⁺², 15%), 648.4 ([M+3H]⁺³, 55%), 486.6 ([M+4H]⁺⁴, 100%).

Example 33 Synthesis of3-(7-[3-(amidinoamino)propyl]-2,5-dioxo-1-{[4-(3-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}propoxy)phenyl]methyl}-3H-benzo[f]1,4-diazaperhydroepin-4-yl)propanoicacid

Example 34 Synthesis of3-(8-[3-(amidinoamino)propyl]-2,5-dioxo-1-{[4-(3-{2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]acetylamino}propoxy)phenyl]methyl)-3H-benzo[f]1,4-diazaperhydroepin-4-yl)propanoicacid

Compounds in Example 33 and Example 34 are prepared by the syntheticroute shown in Scheme I.

The procedure described below elucidates Scheme I:

-   Step I: 7-Substituted or 8-substituted IB may be prepared via the    alkylation of ethyl    3-(7-(3-[(tert-butoxy)carbonylamino]propyl}-2,5-dioxo-1H,3H-benzo[f]1,4-diazaperhydroepin-4-yl)propanoate    or ethyl    3-(8-{3-[(tert-butoxy)carbonylamino]propyl}-2,5-dioxo-1H,3H-benzo[f]1,4-diazaperhydroepin-4-yl)propanoate    (IA) with 4-(2,4-dimethoxybenzyloxy)benzylbromide in the presence of    base, followed by removal of the 2,4-dimethoxybenzyl protecting    group. Alternately, 7-substituted or 8-substituted IB may be    prepared from 1-aryl-6-iodoisatoic anhydride and    1-aryl-7-iodoisatoic anhydride by methods known in the art    (McDowell, R. S. et al, J. Amer. Chem. Soc., 1994, 116, 5077–5083    and Blackburn, B. et al, PCT Intl. Appl., WO 9308174 Al 19930429    (CAS: 120:217745)).-   Step II: Compound IC is prepared by the alkylation of the hydroxyl    group in IB with 3-Cbz-aminopropylbromide in the presence of base    such as NaH in a solvent such as DMF.-   Step III: Intermediate ID is prepared by the deprotection of the Boc    group with either trifluoroacetic acid or HCl-ethyl acetate followed    by treatment of the intermediate with formamidinosulfonic acid in    the presence of base (eg. 5% KHCO₃).-   Step IV: Removal of the benzyloxycarbonyl group (Z, Cbz) is achieved    by hydrogenolysis (Pd/C) or TFA/triethylsilane to give IE.-   Step V: The title compound IF is prepared by the conjugation of IE    with DO3A-tri-t-butyl ester (Macrocyclics), followed by base and TFA    hydrolyses of the ethyl and t-butyl esters, respectively. The    desired compound is purified by reversed phase preparative HPLC.

UTILITY

The pharmaceuticals of the present invention are useful for imagingangiogenic tumor vasculature, therapeutic cardiovascular angiogenesis,and cardiac pathologies associated with the expression of vitronectinreceptors in a patient or for treating cancer in a patient. Theradiopharmaceuticals of the present invention comprised of a gamma rayor positron emitting isotope are useful for imaging of pathologicalprocesses involving angiogenic neovasculature, including cancer,diabetic retinopathy, macular degeneration, restenosis of blood vesselsafter angioplasty, and wound healing, as well as atherosclerotic plaque,myocardial reperfusion injury, and myocardial ischemia, stunning orinfarction. The radiopharmaceuticals of the present invention comprisedof a beta, alpha or Auger electron emitting isotope are useful fortreatment of pathological processes involving angiogenic neovasculature,by delivering a cytotoxic dose of radiation to the locus of theangiogenic neovasculature. The treatment of cancer is affected by thesystemic administration of the radiopharmaceuticals resulting in acytotoxic radiation dose to tumors.

The compounds of the present invention comprised of one or moreparamagnetic metal ions selected from gadolinium, dysprosium, iron, andmanganese, are useful as contrast agents for magnetic resonance imaging(MRI) of pathological processes involving angiogenic neovasculature, aswell as atherosclerotic plaque, myocardial reperfusion injury, andmyocardial ischemia, stunning or infarction.

The compounds of the present invention comprised of one or more heavyatoms with atomic number of 20 or greater are useful as X-ray contrastagents for X-ray imaging of pathological processes involving angiogenicneovasculature, as well as atherosclerotic plaque, myocardialreperfusion injury, and myocardial ischemia, stunning or infarction.

The compounds of the present invention comprised of an echogenic gascontaining surfactant microsphere are useful as ultrasound contrastagents for sonography of pathological processes involving angiogenicneovasculature, as well as atherosclerotic plaque, myocardialreperfusion injury, and myocardial ischemia, stunning or infarction.

Representative compounds of the present invention were tested in thefollowing in vitro assays and in vivo models and were found to beactive.

Immobilized Human Placental α_(v)β₃ Receptor Assay

The assay conditions were developed and validated using[I-125]vitronectin. Assay validation included Scatchard format analysis(n=3) where receptor number (Bmax) and Kd (affinity) were determined.Assay format is such that compounds are preliminarily screened at 10 and100 nM final concentrations prior to IC₅₀ determination. Three standards(vitronectin, anti-α_(v)β₃ antibody, LM609, and anti-α_(v)β₅, P1F6) andfive reference peptides have been evaluated for IC₅₀ determination.Briefly, the method involves immobilizing previously isolated receptorsin 96 well plates and incubating overnight. The receptors were isolatedfrom normal, fresh, non-infectious (HIV, hepatitis B and C, syphilis,and HTLV free) human placenta. The tissue was lysed and tissue debrisremoved via centrifugation. The lysate was filtered. The receptors wereisolated by affinity chromatography using the immobilized α_(v)β₃antibody. The plates are then washed 3× with wash buffer. Blockingbuffer is added and plates incubated for 120 minutes at roomtemperature. During this time compounds to be tested and[I-125]vitronectin are premixed in a reservoir plate. Blocking buffer isremoved and compound mixture pipetted. Competition is carried out for 60minutes at room temperature. Unbound material is then removed and wellsare separated and counted via gamma scintillation.

Oncomouse® Imaging

The study involves the use of the c-Neu Oncomouse® and FVB micesimultaneously as controls. The mice are anesthetized with sodiumpentobarbital and injected with approximately 0.5 mCi ofradiopharmaceutical. Prior to injection, the tumor locations on eachOncomouse® are recorded and tumor size measured using calipers. Theanimals are positioned on the camera head so as to image the anterior orposterior of the animals. 5 Minute dynamic images are acquired seriallyover 2 hours using a 256×256 matrix and a zoom of 2×. Upon completion ofthe study, the images are evaluated by circumscribing the tumor as thetarget region of interest (ROI) and a background site in the neck areabelow the carotid salivary glands.

This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake in the tumors can bequantified either non-invasively by imaging for those isotopes with acoincident imageable gamma emission, or by excision of the tumors andcounting the amount of radioactivity present by standard techniques. Thetherapeutic effect of the radiopharmaceuticals can be assessed bymonitoring the rate of growth of the tumors in control mice versus thosein the mice administered the radiopharmaceuticals of the presentinvention.

This model can also be used to assess the compounds of the presentinvention comprised of paramagnetic metals as MRI contrast agents. Afteradministration of the appropriate amount of the paramagnetic compounds,the whole animal can be placed in a commercially available magneticresonance imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of heavy atoms as X-ray contrast agents. Afteradministration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the tumors in the animalcan be imaging using an ultrasound probe held proximate to the tumors.The effectiveness of the contrast agents can be readily seen bycomparison to the images obtain from animals that are not administered acontrast agent.

Rabbit Matrigel Model

This model was adapted from a matrigel model intended for the study ofangiogenesis in mice. Matrigel (Becton & Dickinson, USA) is a basementmembrane rich in laminin, collagen IV, entactin, HSPG and other growthfactors. When combined with growth factors such as bFGF [500 ng/ml] orVEGF [2 μg/ml] and injected subcutaneously into the mid-abdominal regionof the mice, it solidifies into a gel and stimulates angiogenesis at thesite of injection within 4–8 days. In the rabbit model, New ZealandWhite rabbits (2.5–3.0 kg) are injected with 2.0 ml of matrigel, plus 1μg bFGF and 4 μg VEGF. The radiopharmaceutical is then injected 7 dayslater and the images obtained.

This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake at the angiogenicsites can be quantified either non-invasively by imaging for thoseisotopes with a coincident imageable gamma emission, or by excision ofthe angiogenic sites and counting the amount of radioactivity present bystandard techniques. The therapeutic effect of the radiopharmaceuticalscan be assessed by monitoring the rate of growth of the angiogenic sitesin control rabbits versus those in the rabbits administered theradiopharmaceuticals of the present invention.

This model can also be used to assess the compounds of the presentinvention comprised of paramagnetic metals as MRI contrast agents. Afteradministration of the appropriate amount of the paramagnetic compounds,the whole animal can be placed in a commercially available magneticresonance imager to image the angiogenic sites. The effectiveness of thecontrast agents can be readily seen by comparison to the images obtainfrom animals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of heavy atoms as X-ray contrast agents. Afteradministration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the angiogenic sites. The effectiveness of thecontrast agents can be readily seen by comparison to the images obtainfrom animals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the angiogenic sites inthe animal can be imaging using an ultrasound probe held proximate tothe tumors. The effectiveness of the contrast agents can be readily seenby comparison to the images obtain from animals that are notadministered a contrast agent.

Canine Spontaneous Tumor Model

Adult dogs with spontaneous mammary tumors were sedated with xylazine(20 mg/kg)/atropine (1 ml/kg). Upon sedation the animals were intubatedusing ketamine (5 mg/kg)/diazepam (0.25 mg/kg) for full anethesia.Chemical restraint was continued with ketamine (3 mg/kg)/xylazine (6mg/kg) titrating as necessary. If required the animals were ventilatedwith room air via an endotrachael tube (12 strokes/min, 25 ml/kg) duringthe study. Peripheral veins were catheterized using 20G I.V. catheters,one to serve as an infusion port for compound while the other forexfusion of blood samples. Heart rate and EKG were monitored using acardiotachometer (Biotech, Grass Quincy, Mass.) triggered from a lead IIelectrocardiogram generated by limb leads. Blood samples are generallytaken at ˜10 minutes (control), end of infusion, (1 minute), 15 min, 30min, 60 min, 90 min, and 120 min for whole blood cell number andcounting. Radiopharmaceutical dose was 300 uCi/kg adminitered as an i.v.bolus with saline flush. Parameters were monitored continuously on apolygraph recorder (Model 7E Grass) at a paper speed of 10 mm/min or 10mm/sec.

Imaging of the laterals were for 2 hours with a 256×256 matrix, no zoom,5 minute dynamic images. A known source is placed in the image field(20–90 μCi) to evaluate region of interest (ROI) uptake. Images werealso acquired 24 hours post injection to determine retention of thecompound in the tumor. The uptake is determined by taking the fractionof the total counts in an inscribed area for ROI/source and multiplyingthe known μCi. The result is μCi for the ROI.

This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake in the tumors can bequantified either non-invasively by imaging for those isotopes with acoincident imageable gamma emission, or by excision of the tumors andcounting the amount of radioactivity present by standard techniques. Thetherapeutic effect of the radiopharmaceuticals can be assessed bymonitoring the size of the tumors over time.

This model can also be used to assess the compounds of the presentinvention comprised of paramagnetic metals as MRI contrast agents. Afteradministration of the appropriate amount of the paramagnetic compounds,the whole animal can be placed in a commercially available magneticresonance imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of heavy atoms as X-ray contrast agents. Afteradministration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the tumors in the animalcan be imaging using an ultrasound probe held proximate to the tumors.The effectiveness of the contrast agents can be readily seen bycomparison to the images obtain from animals that are not administered acontrast agent.

Cardiovascular disease models that can be used to assess the diagnosticradiopharmaceuticals, magnetic resonance, X-ray and ultrasound contrastagents of the present invention are reviewed in J. Nucl. Cardiol., 1998,5, 167–83. There are several well established rabbit models ofatherosclerosis; one model produces predominantly proliferating smoothmuscle cells by balloon deendothelialization of infradiaphragmaticabdominal aorta to simulate restenotic lesions; another model thatproduces simulated advanced human atherosclerotic plaque by balloondeendothelialization followed by a high cholesterol diet.

A model of congestive heart failure is described in Am. J. Physiol.,1998, 274, H1516-23. In general, Yorkshire pigs are randomly assigned toundergo 3 wks of rapid atrial pacing at 240 beats/min. or to be shamcontrols. The pigs are chronically instrumented to measure leftventricular function in the conscious state. The pigs are anesthetized.A shielded stimulating electrode is sutured onto the left atrium,connected to a modified programmable pace maker and buried in asubcutaneous pocket. The pericardium is closed loosely, the thoracotomyis closed, and the pleural space is evacuated of air. After a recoveryperiod of 7–10 days, the pacemaker is activated in the animals selectedto undergo chronic rapid pacing. The animals are sedated, the pacemakeris deactivated (pacing groups only. After a 30 min stabilization period,indexes of LV function and geometry are determined (by echocardiographyas a control) by injecting the radiolabeled compound. Forbiodistribution, the animals are anesthetized, the heart extirpate andthe LV apex and midventricular regions are evaluated.

A rat model of reversible coronary occlusion and reperfusion isdescribed in McNulty et al., J. Am. Physiol., 1996, H2283-9.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise that as specifically describedherein.

1. A diagnostic or therapeutic metallopharmaceutical compositioncomprising: at least one agent selected from the group consisting of achemotherapeutic agent and a radiosensitizer agent, or apharmaceutically acceptable salt thereof, and a radiopharmaceuticalcomprising a metal and a compound comprising a targeting moiety bound toa chelator; wherein the targeting moiety is bound to the chelator by 0–1linking groups, and wherein the targeting moiety is a benzodiazepine,benzodiazepinedione, or dibenzotrihydroannulene nonpeptide, and binds toa receptor that is upregulated during angiogenesis.
 2. Themetallopharmaceutical composition of claim 1, wherein thechemotherapeutic agent is selected from the group consisting ofmitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide,cladribine, mitobronitol, methotrexate, doxorubicin, carboquone,pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed,daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane,nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone,aminoglutethimide, amsacrine, proglumide, elliptinium acetate,ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin,nimustine, vindesine, flutamide, drogenil, butocin, carmoflir, razoxane,sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine,picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride,oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol,formestane, interferon-alpha, interferon-2 alpha, interferon-beta,interferon-gamma, colony stimulating factor-1, colony stimulatingfactor-2, denileukin diftitox, interleukin-2, and leutinizing hormonereleasing factor.
 3. The metallopharmaceutical composition of claim 1,wherein radiosensitizer agent is selected from the group consisting of2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,3-amino-1,2,4-benzotriazine-1,4-dioxide,N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.
 4. Themetallopharmaceutical composition of claim 1, wherein the metal is^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ³³P, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La,¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, ¹⁹²Ir,Gd(III), Dy(III), Fe(III), or Mn(II).
 5. The metallopharmaceuticalcomposition of claim 1, wherein the targeting moiety is a compound ofFormula (Ia):

wherein: R¹ and R³ are independently selected from the group: C₁–C₆alkyl, benzyl, phenethyl, and a bond to the linking group; provided thatone of R¹ and R³ is a bond to the linking group; R² is independentlyselected from the group: 2-benzimidazolylmethyl, 2-guanidinoethyl,2-amino-2-pyridyl, 2-amino-2-pyridylmethyl, 5-amino-2-imidazolylmethyl,and 2-imidazolylmethyl; R⁴ is independently selected from H, C₁₋₆ alkylor benzyl.
 6. The metallopharmaceutical composition of claim 1, whereinthe targeting moiety is a compound of Formula (Ib):

wherein: R^(2a) is (CH₂)₃R^(3a); R^(3a) is selected from the group:

R^(4a) is independently selected from C₁₋₆ alkyl substituted with a bondto the linking group or benzyl substituted with a bond to the linkinggroup.
 7. The metallopharmaceutical composition of claim 1, wherein thetargeting moiety is a compound of Formula (Ic):

wherein: R^(2b) is independently selected from the group:

wherein the asterisks * denote optional positions for attaching to thelinking group.
 8. The metallopharmaceutical composition of claim 1,further comprising an additional targeting moiety that is a peptideselected from the group:

wherein: R^(1p) is L-valine, D-valine or L-lysine optionally substitutedon the ε amino group with a bond to the linking group; R^(2p) isL-phenylalanine, D-phenylalanine, D-1-naphthylalanine,2-aminothiazole-4-acetic acid or tyrosine, the tyrosine optionallysubstituted on the hydroxy group with a bond to the linking group;R^(3p) is D-valine; R^(4p) is D-tyrosine substituted on the hydroxygroup with a bond to the linking group; provided that one of R^(1p) andR^(2p) in each Q is substituted with a bond to the linking group, andfurther provided that when R^(2p) is 2-aminothiazole-4-acetic acid, K isN-methylarginine.
 9. The metallopharmaceutical composition of claim 1,wherein the chelator is a metal bonding unit having a formula selectedfrom the group:

A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at eachoccurrence from the group: NR¹³, NR¹³R¹⁴, S, SH, S(Pg), O, OH, PR¹³,PR¹³R¹⁴, P(O)R¹⁵R¹⁶, and a bond to the linking group; E is a bond, CH,or a spacer group independently selected at each occurrence from thegroup: C1–C10 alkyl substituted with 0–3 R¹⁷, aryl substituted with 0–3R¹⁷, C3–10 cycloalkyl substituted with 0–3 R¹⁷, heterocyclo-C1–10 alkylsubstituted with 0–3 R¹⁷, wherein the heterocyclo group is a 5–10membered heterocyclic ring system containing 1–4 heteroatomsindependently selected from N, S, and O, C6–10 aryl-C1–10 alkylsubstituted with 0–3 R¹⁷, C1–10 alkyl-C6–10 aryl-substituted with 0–3R¹⁷, and a 5–10 membered heterocyclic ring system containing 1–4heteroatoms independently selected from N, S, and O and substituted with0–3 R¹⁷; R¹³ and R¹⁴ are each independently selected from the group: abond to the linking group, hydrogen, C1–C10 alkyl substituted with 0–3R¹⁷, aryl substituted with 0–3 R¹⁷, C1–10 cycloalkyl substituted with0–3 R¹⁷, heterocyclo-C1–10 alkyl substituted with 0–3 R¹⁷, wherein theheterocyclo group is a 5–10 membered heterocyclic ring system containing1–4 heteroatoms independently selected from N, S, and O, C6–10aryl-C1–10 alkyl substituted with 0–3 R¹⁷, C1–10 alkyl-C6–10aryl-substituted with 0–3 R¹⁷, a 5–10 membered heterocyclic ring systemcontaining 1–4 heteroatoms independently selected from N, S, and O andsubstituted with 0–3 R¹⁷, and an electron, provided that when one of R¹³or R¹⁴ is an electron, then the other is also an electron;alternatively, R¹³ and R¹⁴ combine to form ═C(R²⁰)(R²¹); R¹⁵ and R¹⁶ areeach independently selected from the group: a bond to the linking group,—OH, C1–C10 alkyl substituted with 0–3 R¹⁷, C1–C10 alkyl substitutedwith 0–3 R¹⁷, aryl substituted with 0–3 R¹⁷, C3–10 cycloalkylsubstituted with 0–3 R¹⁷, heterocyclo-C1–10 alkyl substituted with 0–3R¹⁷, wherein the heterocyclo group is a 5–10 membered heterocyclic ringsystem containing 1–4 heteroatoms independently selected from N, S, andO, C6–10 aryl-C1–10 alkyl substituted with 0–3 R¹⁷, C1–10 alkyl-C6–10aryl-substituted with 0–3 R¹⁷, and a 5–10 membered heterocyclic ringsystem containing 1–4 heteroatoms independently selected from N, S, andO and substituted with 0–3 R¹⁷; R¹⁷ is independently selected at eachoccurrence from the group: a bond to the linking group, ═O, F, Cl, Br,I, —CF₃, —CN, —CO₂R¹⁸, —C(═O)R¹⁸, —C(O)N(R¹⁸)₂, —CHO, —CH₂OR¹⁸,—OC(═O)R¹⁸, —OC(═O)OR^(18a), —OR¹⁸, —OC(═O)N(R¹⁸)₂, —NR¹⁹C(═O)R¹⁸,—NR¹⁹C(═O)OR^(18a), —NR¹⁹C(═O)N(R¹⁸)₂, —NR¹⁹SO₂N(R¹⁸)₂, —NR¹⁹SO₂R^(18a),—SO₃H, —SO₂R^(18a), —SR¹⁸, —S(═O)R¹⁸, —SO₂N(R¹⁸)₂, —N(R¹⁸)₂,—NHC(═S)NHR¹⁸, ═NOR¹⁸, NO₂, —C(═O)NHOR¹⁸, —C(═O)NHNR¹⁸R^(18a),—OCH₂CO₂H, 2-(1-morpholino)ethoxy, C1–C5 alkyl, C2–C4 alkenyl, C3–C6cycloalkyl, C3–C6 cycloalkylmethyl, C2–C6 alkoxyalkyl, aryl substitutedwith 0–2 R¹⁸, and a 5–10 membered heterocyclic ring system containing1–4 heteroatoms independently selected from N, S, and O; R¹⁸, R^(18a),and R¹⁹ are independently selected at each occurrence from the group: abond to the linking group, H, C1–C6 alkyl, phenyl, benzyl, C1–C6 alkoxy,halide, nitro, cyano, and trifluoromethyl; Pg is a thiol protectinggroup; R²⁰ and R²¹ are independently selected from the group: H, C₁–C₁₀alkyl, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, C₂–C₁₀ 1-alkynesubstituted with 0–3 R²³, C₂–C₁₀ 1-alkyne substituted with 0–3 R²³, arylsubstituted with 0–3 R²³, unsaturated 5–10 membered heterocyclic ringsystem containing 1–4 heteroatoms independently selected from N, S, andO and substituted with 0–3 R²³, and unsaturated C₃₋₁₀ carbocyclesubstituted with 0–3 R²³; alternatively, R²⁰ and R²¹, taken togetherwith the divalent carbon radical to which they are attached form:

R²² and R²³ are independently selected from the group: H, R²⁴, C₁–C₁₀alkyl substituted with 0–3 R²⁴, C₂–C₁₀ alkenyl substituted with 0–3 R²⁴,C₂–C₁₀ alkynyl substituted with 0–3 R²⁴, aryl substituted with 0–3 R²⁴,a 5–10 membered heterocyclic ring system containing 1–4 heteroatomsindependently selected from N, S, and O and substituted with 0–3 R²⁴,and C₃₋₁₀ carbocycle substituted with 0–3 R²⁴; alternatively, R²², R²³taken together form a fused aromatic or a 5–10 membered heterocyclicring system containing 1–4 heteroatoms independently selected from N, S,and O; a and b indicate the positions of optional double bonds and n is0 or 1; R²⁴ is independently selected at each occurrence from the group:═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R²⁵, —C(═O)R²⁵, —C(═O)N(R²⁵)₂, —N(R²⁵)₃⁺, —CH₂OR²⁵, —OC(═O)R²⁵, —OC(═O)OR²⁵a, —OR²⁵, —OC(═O)N(R²⁵)₂,—NR²⁶C(═O)R²⁵, —NR²⁶C(═O)OR^(25a), —NR²⁶C(═O)N(R²⁵)₂, —NR²⁶SO₂N(R²⁵)₂,—NR²⁶SO₂R^(25a), —SO₃H, —SO₂R^(25a), —SR²⁵, —S(═O)R^(25a), —SO₂N(R²⁵)₂,—N(R²⁵)₂, ═NOR²⁵, —C(═O)NHOR²⁵, —OCH₂CO₂H, and 2-(1-morpholino)ethoxy;and, R²⁵, R^(25a), and R²⁶ are each independently selected at eachoccurrence from the group: hydrogen and C1–C6 alkyl.
 10. Themetallopharmaceutical composition of claim 1, wherein the linking groupis present between the targeting moiety and chelator.
 11. Themetallopharmaceutical composition of claim 1, wherein the linking grouphas the formula:((W)_(h)—(CR⁶R⁷)_(g))_(x)-(Z)_(k)-((CR^(6a)R^(7a))_(g′)—(W)_(h′))_(x′)wherein: W is independently selected at each occurrence from the group:O, S, NH, NHC(═O), C(═O)NH, NR⁸C(═O), C(═O)NR⁸, C(═O), C(═O)O, OC(═O),NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′),(OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′); aa is independentlyat each occurrence an amino acid; Z is selected from the group: arylsubstituted with 0–3 R¹⁰, C3–10 cycloalkyl substituted with 0–3 R¹⁰, anda 5–10 membered heterocyclic ring system containing 1–4 heteroatomsindependently selected from N, S, and O and substituted with 0–3 R¹⁰;R⁶, R^(6a), R⁷, R^(7a), and R⁸ are independently selected at eachoccurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C1–C5 alkylsubstituted with 0–3 R¹⁰, aryl substituted with 0–3 R¹⁰, benzylsubstituted with 0–3 R¹⁰, and C1–C5 alkoxy substituted with 0–3 R¹⁰,NHC(═O)R¹¹, C(═O)NHR¹¹, NHC(═O)NHR¹¹, NHR¹¹, R¹¹, and a bond to thechelator; R¹⁰ is independently selected at each occurrence from thegroup: a bond to the chelator, COOR¹¹, C(═O)NHR¹¹, NHC(═O)R¹¹, OH,NHR¹¹, SO₃H, PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0–3 R¹¹, C1–5alkyl substituted with 0–1 R¹², C1–5 alkoxy substituted with 0–1 R¹²,and a 5–10 membered heterocyclic ring system containing 1–4 heteroatomsindependently selected from N, S, and O and substituted with 0–3 R¹¹;R¹¹ is independently selected at each occurrence from the group: H,alkyl substituted with 0–1 R¹², aryl substituted with 0–1 R¹², a 5–10membered heterocyclic ring system containing 1–4 heteroatomsindependently selected from N, S, and O and substituted with 0–1 R¹²,C3–10 cycloalkyl substituted with 0–1 R¹², polyalkylene glycolsubstituted with 0–1 R¹², carbohydrate substituted with 0–1 R¹²,cyclodextrin substituted with 0–1 R¹², amino acid substituted with 0–1R¹², polycarboxyalkyl substituted with 0–1 R¹², polyazaalkyl substitutedwith 0–1 R¹², peptide substituted with 0–1 R¹², wherein the peptide iscomprised of 2–10 amino acids, 3,6-O-disulfo-B-D-galactopyranosyl,bis(phosphonomethyl)glycine, and a bond to the chelator; R¹² is a bondto the chelator; k is selected from 0, 1, and 2; h is selected from 0,1, and 2; h′ is selected from 0, 1, and 2; g is selected from 0, 1, 2,3, 4, 5, 6, 7, 8, 9, and 10; g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7,8, 9, and 10; s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; s″ is selectedfrom 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; t is selected from 0, 1,2, 3,4, 5, 6, 7, 8, 9, and 10; t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8,9, and 10; x is selected from 0, 1, 2, 3, 4, and 5; and x′ is selectedfrom 0, 1, 2, 3, 4, and
 5. 12. The metallopharmaceutical composition ofclaim 1, wherein the receptor is α_(v)β₃ or α_(v)β₅.
 13. A compositioncomprising: a compound comprising a benzodiazepine, benzodiazepinedione,or dibenzotrihydroannulene nonpeptide targeting moiety bound to achelator, optionally through a linking group, wherein the targetingmoiety binds to a receptor that is upregulated during angiogenesis; ametal; and at least one of a chemotherapeutic agent or a radiosensitizeragent; or a pharmaceutically acceptable salt thereof.
 14. A compositioncomprising: a compound comprising a benzodiazepine, benzodiazepinedione,or dibenzotrihydroannulene nonpeptide targeting moiety bound to achelator, optionally through a linking group, wherein the targetingmoiety binds to a receptor that is upregulated during angiogenesis; ametal; and at least one of mitomycin, tretinoin, ribomustin,gemcitabine, vincristine, etoposide, cladribine, mitobronitol,methotrexate, doxorubicin, carboquone, pentostatin, nitracrine,zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole,fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine,bicalutamide, vinorelbine, vesnarmnone, aminoglutethimide, amsacrine,proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate,isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil,butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol,tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide,improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen,progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha,interferon-2 alpha, interferon-beta, interferon-gamma, colonystimulating factor-i, colony stimulating factor-2, denileukin diftitox,interleukin-2, leutinizing hormone releasing factor,2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,3-amino-1,2,4-benzotriazine-1,4-dioxide,N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, or1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol; or apharmaceutically acceptable salt thereof.